Podophyllotoxin Derivatives

ABSTRACT

4-O esters of podophyllotoxin and 4′-demethylepipodophyllotoxin are provided. The compounds are 4-O esters of an alkanoic acid or substituted alkanoic acid and podophyllotoxin and 4′-demethylepipodophyllotoxin. The compounds are useful for treating cancer.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a Divisional Application of the copending U.S.Utility patent application Ser. No. 10/612,240, filed Jul. 1, 2003,which is hereby incorporated by reference in its entity.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY-SPONSOREDRESEARCH AND DEVELOPMENT

The U.S. Government has a paid-up license in this invention and theright in limited circumstances to require the patent owner to licenseothers on reasonable terms as provided for by the terms of grant numberDAMD-99-1-9018, awarded by the U.S. Army Medical Research AcquisitionActivity.

INTRODUCTION

1. Field of the Invention

This invention relates to novel derivatives of podophyllotoxin that areuseful for treating various types of cancer.

2. Background of the Invention

Podophyllotoxin is a known compound having the formula:

The compound shows activity as an antiviral and as an antineoplasticagent. This invention relates to novel derivatives of the compound thatare useful for treating cancer.

SUMMARY OF THE INVENTION

One aspect of the invention is a compound represented by the formula:

where R is C(O)—(CH₂)_(m)—X—R₁, wherein m is 0-10, X is S, O, N or acovalent bond, and R₁ is optionally substituted phenyl, optionallysubstituted cycloalkyl having 3 to 7 carbons forming the ring,optionally substituted fused 2-, 3-, or 4-ring heterocycle, optionallysubstituted 1- or 2-naphthyl, optionally substituted 5- or 6-memberedheterocycle, optionally substituted anthraquinone, or hemisuccinic acid,with the proviso that when m is 0 and X is a bond, R₁ cannot be phenylor substituted phenyl; when X is a bond and R₁ is phenyl, m cannot be 2and when X is O, m cannot be 1.

Another aspect of the invention is a compound represented by theformula:

where R is C(O)—(CH₂)_(m)—X—R₁, wherein m is 0-10, X is S, O, N or acovalent bond, and R₁ is optionally substituted phenyl, optionallysubstituted cycloalkyl having 3 to 7 carbons forming the ring,optionally substituted fused 2-, 3-, or 4-ring heterocycle, optionallysubstituted 1- or 2-naphthyl, optionally substituted 5- or 6-memberedheterocycle, optionally substituted anthraquinone, hemisuccinic acid;and R₂ is hydrogen, PO₃H₂ or PO(OR₃)₂ where R₃ is benzyl.

Another aspect of this invention is a compound of the formula A-R₅—Bwherein each of A and B independently is represented by the radical

wherein R₂ is hydrogen, PO₃H₂ or PO(OR₃)₂ where R₃ is benzyl and R₅ is adicarboxy linker.

Another aspect of the invention is a pharmaceutical composition usefulfor treating cancer in a warm-blooded animal, which compositioncomprises compound of the invention as defined herein in combinationwith a pharmaceutically acceptable excipient.

Another aspect of this invention is a method for treating cancer in awarm-blooded animal, which method comprises administering atherapeutically effective amount of a compound of the invention asdefined herein. The compound is administered in a therapeuticallyeffective dose by appropriate administration, e.g. orally, topically, orparenterally.

Another aspect of this invention is a process for preparing compounds ofthis invention by reacting podophyllotoxin (PT) or4′-demethylepipodophyllotoxin (DPT) with a compound of the formulaYC(O)(CH₂)_(m)XR₁, wherein m, X, and R₁, are as defined herein, and Y ise.g. bromide, chloride, hydroxy, or alkoxy. Preferably Y is OH.

Other aspects of this invention will be apparent to one of skill in theart by reviewing the ensuing specification.

DETAILED DESCRIPTION

Overview

In general this invention can be viewed as derivatives ofpodophyllotoxin or 4′-demethylepipodophyllotoxin. The novel compounds ofthe invention are active against tumors in mice and are generally welltolerated. They are useful for treating various types of cancer and canbe formulated to prepare pharmaceutical preparations, e.g. for oral,topical, or parenteral administration.

While not wishing to be bound by any particular mechanism of action ortheoretical explanation of how the compounds work, it is believed thatthe principal mechanism of action of the compounds of the invention isthe inhibition of the catalytic activity of type II DNA topoisomerase(topoisomerase II) and concurrent enzyme-mediated production of lethalDNA strand breaks.

DEFINITIONS

The term “alkyl” refers to a monovalent, saturated aliphatic hydrocarbonradical having the indicated number of carbon atoms. For example, a “C1-6 alkyl” or an “alkyl of 1-6 carbons” or “Alk 1-6” would refer to anyalkyl group containing one to six carbons in the structure. “C 1-20alkyl” refers to any alkyl group having one to twenty carbons. Alkyl maybe a straight chain (i.e. linear) or a branched chain. Lower alkylrefers to an alkyl of 1-6 carbons. Representative examples lower alkylradicals include methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl,isopropyl, isobutyl, isopentyl, amyl, sec-butyl, tert-butyl, tert-pentyland the like. Higher alkyl refers to alkyls of seven carbons and above.These include n-heptyl, n-octyl, n-nonyl, n-decyl, n-dodecyl,n-tetradecyl, n-hexadecyl, n-octadecyl, n-eicosyl, and the like, alongwith branched variations thereof. The radical may be optionallysubstituted with substituents at positions that do not significantlyinterfere with the preparation of compounds falling within the scope ofthis invention and that do not significantly reduce the efficacy of thecompounds. The alkyl may be optionally substituted with one to fivesubstituents independently selected from the group consisting of halo,lower alkoxy, hydroxy, cyano, nitro, or amino.

The term “alkoxy” refers to a monovalent radical of the formula RO—,where R is an alkyl as defined herein. Lower alkoxy refers to an alkoxyof 1-6 carbon atoms, with higher alkoxy is an alkoxy of seven or morecarbon atoms. Representative lower alkoxy radicals include methoxy,ethoxy, n-propoxy, n-butoxy, n-pentyloxy, n-hexyloxy, isopropoxy,isobutoxy, isopentyloxy, amyloxy, sec-butoxy, tert-butoxy,tert-pentyloxy, and the like. Higher alkoxy radicals include thosecorresponding to the higher alkyl radicals set forth herein. The radicalmay be optionally substituted with substituents at positions that do notsignificantly interfere with the preparation of compounds falling withinthe scope of this invention and that do not significantly reduce theefficacy of the compounds. The radical may be optionally substitutedwith one to five substituents independently selected from the groupconsisting of halo, lower alkyl, lower alkoxy, hydroxy, cyano, nitro, oramino.

The term “cycloalkyl” refers to a monovalent, alicyclic, saturatedhydrocarbon radical having three or more carbons forming the ring. Whileknown cycloalkyl compounds may have up to 30 or more carbon atoms,generally there will be three to seven carbons in the ring. The latterinclude, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,and cycloheptyl. The radical may be optionally substituted withsubstituents at positions that do not significantly interfere with thepreparation of compounds falling within the scope of this invention andthat do not significantly reduce the efficacy of the compounds. Thecycloalkyl is optionally substituted with one to five substituentsindependently selected from the group consisting of halo, lower alkyl,lower alkoxy, hydroxy, cyano, nitro, amino, halogenated lower alkyl,halogenated lower alkoxy, hydroxycarbonyl, lower alkoxycarbonyl, loweralkylcarbonyloxy, and lower alkylcarbonylamino.

The term “hydroxycarbonyl” is a monovalent radical having the formula—C(O)OH.

The term “lower alkoxycarbonyl” is a monovalent radical having theformula —C(O)OAlk, where Alk is lower alkyl.

The term “lower alkylcarboxyloxy” is a monovalent radical having theformula —OC(O)Alk, where Alk is lower alkyl.

The term “lower alkylcarbonylamino” is a monovalent radical having theformula —NHC(O)Alk, where Alk is lower alkyl.

The term “alkylamino” is a monovalent radical having the formula —NR₁,R₂ where R₁ is alkyl and R₂ is hydrogen or alkyl and the alkyl isoptionally substituted.

A “halo” substituent is a monovalent halogen radical chosen from chloro,bromo, iodo, and fluoro. A “halogenated” compound is one substitutedwith one or more halo substituent.

A “phenyl” is a radical formed by removal of a hydrogen from a benzenering. The phenyl is optionally substituted with from one to fivesubstituents independently selected from the group consisting of halo,lower alkyl, lower alkoxy, hydroxy, cyano, nitro, amino, halogenatedlower alkyl, halogenated lower alkoxy, carbonyl, hydroxycarbonyl, loweralkylcarbonyloxy, benzyloxy, optionally substituted piperidino, loweralkoxycarbonyl, and lower alkylcarbonylamino. The phenyl may also besubstituted by a camptothecin or a camptothecin derivative through acarbonyl group attached to the E ring of the camptothecin at the 20S-oxygen. Such compound are known in the art, see, for example, U.S.Pat. No. 6,350,756 or 6,403,604, which incorporated herein by referencein their entirety.

A “dicarboxy linker” is a divalent radical having two carboxy groups(C(O)—) that link two molecules such as podophyllotoxin or4′-demethylepipodophyllotoxin together at an oxygen linkage, e.g., the4-position of the podophyllotoxin molecule. Such linkers includestraight chain or cyclic linkers and include by way of example5-nitroisophthalic acid and 3,5 pyridine dicarboxylic acid.

A “carbamoyloxy” is a monovalent radical of the formula R₁₃R₁₄NC(O)O—(i.e. an aminocarbonyloxy) where R₁₃ and R₁₄ together form a cyclicamino with the nitrogen atom, or each of R₁₃ and R₁₄ is independentlyhydrogen, lower alkyl, hydroxy lower alkyl, hydroxy lower alkyl, aminolower alkyl, lower cycloalkyl, phenyl (substituted or unsubstituted), orbenzyl (substituted or unsubstituted). Examples includeaminocarbonyloxy, methylaminocarbonyloxy, dimethyl aminocarbonyloxy,[4-(1-piperidino)-1-piperidino]carbonyloxy, 1-morpholinocarbonyloxy,1-pyrrolidinyl, 1-piperazinecarbonyloxy, and others delineated herein.

A “5-membered heterocyclic ring” is a monovalent radical of a 5-memberclosed ring containing carbon and at least one other element, generallynitrogen, oxygen, or sulfur and may be fully saturated, partiallysaturated, or unsaturated (i.e. aromatic in nature). Generally theheterocycle will contain no more than two hetero atoms. Representativeexamples of unsaturated 5-membered heterocycles with only one heteroatom include 2- or 3-pyrrolyl, 2- or 3-furanyl, and 2- or 3-thiophenyl.Corresponding partially saturated or fully saturated radicals include3-pyrrolin-2-yl, 2- or 3-pyrrolidinyl, 2- or 3-tetrahydrofuranyl, and 2-or 3-tetrahydrothiophenyl. Representative unsaturated 5-memberedheterocyclic radicals having two hetero atoms include imidazolyl,oxazolyl, thiazolyl, pyrazolyl, and the like. The corresponding fullysaturated and partially saturated radicals are also included. Theheterocyclic radical is bonded through an available carbon atom in theheterocyclic ring. The radical may be optionally substituted withsubstituents at positions that do not significantly interfere with thepreparation of compounds falling within the scope of this invention andthat do not significantly reduce the efficacy of the compounds. The ringis optionally substituted with one or two substituents selected from thegroup consisting of halo, lower alkyl, lower alkoxy, hydroxy, cyano,nitro, amino, halogenated lower alkyl, halogenated lower alkoxy,hydroxycarbonyl, lower alkoxycarbonyl, lower alkylcarbonyloxy, and loweralkylcarbonylamino. Excluded from the definition is a compound that is acarbocyclic imide compound such as succinimide or a hemi-succinimide.

A “6-membered heterocyclic ring” is a monovalent radical of a 6-memberclosed ring containing carbon and at least one other element, generallynitrogen, oxygen, or sulfur and may be fully saturated, partiallysaturated, or unsaturated (i.e. aromatic in nature). Generally theheterocycle will contain no more than two hetero atoms. Representativeexamples of unsaturated 6-membered heterocycles with only one heteroatom include 2-, 3-, or 4-pyridinyl, 2H-pyranyl, and 4H-pyranyl.Corresponding partially saturated or fully saturated radicals include2-, 3-, or 4-piperidinyl, 2-, 3-, or 4-tetrahydropyranyl and the like.Representative unsaturated 6-membered heterocyclic radicals having twohetero atoms include 3- or 4-pyridazinyl, 2-, 4-, or 5-pyrimidinyl,2-pyrazinyl, and the like. The corresponding fully saturated andpartially saturated radicals are also included, e.g. 2-piperazine. Theheterocyclic radical is bonded through an available carbon atom in theheterocyclic ring. The radical may be optionally substituted withsubstituents at positions that do not significantly interfere with thepreparation of compounds falling within the scope of this invention andthat do not significantly reduce the efficacy of the compounds. The ringis optionally substituted with one or two substituents selected from thegroup consisting of halo, lower alkyl, lower alkoxy, hydroxy, cyano,nitro, amino, halogenated lower alkyl, halogenated lower alkoxy,hydroxycarbonyl, lower alkoxycarbonyl, lower alkylcarbonyloxy, and loweralkylcarbonylamino. Excluded from the definition is a compound that is acyclic imide compound such as succinimide or a hemi-succinimide.

The term “2-, 3- or 4-ring fused heterocycle” is a 5-, 6-, or 7-memberedheterocyclic ring fused to another, carbocyclic ring or rings or anothersuch 5-, 6-, or 7-membered heterocyclic ring. Representative examplesinclude chromone, quinoline, 1,2,3,4-tetrahydro-carboline,1,2,3,4-tetrahydroisoquinoline, benzofuran, and the like.

A “cyclic amino” is a monovalent radical of a saturated 5-, 6-, or7-membered cyclic amine ring having no more than one additional heteroatom such as nitrogen, oxygen, or sulfur. Representative examplesinclude, e.g., 1-pyrrolidino, 1-piperidino, morpholino, piperazino,3-benzylpiperidino, and the like. These may be substituted orunsubstituted. If substituted, generally they will have no more than 2substituents chosen from lower alkyl, lower cycloalkyl, hydroxy loweralkyl, phenyl (substituted or unsubstituted), benzyl (substituted orunsubstituted), aminocarbonylmethyl, lower alkylaminocarbonylmethyl,amino, mono- or di-lower alkylamino, cyclic amino, or a 5- or 6-memberedheterocyclic ring.

An “imide ring” is a cyclic imide wherein the nitrogen of the cyclicstructure is bonded on each side to a carbonyl group, which in turn isbound to carbon atoms to form a ring. An aromatic fused imide ring wouldinclude, e.g. phthalimide (which may be substituted on the benzenering), 1,8-naphthalimide (which may be substituted on the naphthylring—e.g. 3-nitro-1,8-naphthalimide, 4-nitronaphthalimide,4-bromo-naphthalimide, and the like). Others will be apparent to one ofskill in the art. A carbocyclic imide would include maleimide,succinimide, hemisuccinimide, and the like.

Other chemical terms are given their standard meaning as understood byone of skill in the art with guidance from standard texts anddictionaries.

The term “MTD” is the abbreviation for maximum tolerated does.

The term “nM” is the abbreviation for nanomolar.

The term “ip” is the abbreviation for intraperitoneal.

Compounds of the Invention

One aspect of the invention is a compound represented by the formula(I):

where R is C(O)—(CH₂)_(m)—X—R₁, wherein m is 0-10, X is S, O, N or acovalent bond, and R₁ is optionally substituted phenyl, optionallysubstituted cycloalkyl having 3 to 7 carbons forming the ring,optionally substituted fused 2-, 3-, or 4-ring heterocycle, optionallysubstituted 1- or 2-naphthyl, optionally substituted 5- or 6-memberedheterocycle, optionally substituted anthraquinone, or hemisuccinic acid;with the proviso that when m is 0 and X is a bond, R₁ cannot be phenylor substituted phenyl; when X is a bond and R₁ is phenyl, m cannot be 2and when X is O, m cannot be 1.

Another aspect of the invention is a compound represented by the formula(II):

where R is C(O)—(CH₂)_(m)—X—R₁, wherein m is 0-10, X is S, O, N or acovalent bond, and R₁ is optionally substituted phenyl, optionallysubstituted cycloalkyl having 3 to 7 carbons forming the ring,optionally substituted fused 2-, 3-, or 4-ring heterocycle, optionallysubstituted 1- or 2-naphthyl, optionally substituted 5- or 6-memberedheterocycle, optionally substituted anthraquinone, hemisuccinic acid;and R₂ is hydrogen, PO₃H₂ or PO(OR₃)₂ where R₃ is benzyl.

Another aspect of this invention is a compound of the formula A-R₅—Bwherein each of A and B independently is represented by the radical

wherein R₂ is hydrogen, PO₃H₂ or PO(OR₃)₂ where R₃ is benzyl and R₅ is adicarboxy linker.

A preferred aspect is a compound of formula (I) or formula (II) whereinm is 1-10 and R₁ is phenyl substituted with one to five substituentsindependently selected from halo, lower alkyl, hydroxy, lower alkoxy,cyano, nitro, amino, lower alkylamino, halogenated lower alkylamino,halogenated lower alkyl, halogenated lower alkoxy, carbonyl,hydroxycarbonyl, lower alkylcarbonyloxy, benzyloxy, optionallysubstituted 5- or 6-membered heterocyclic ring, an imide ring, loweralkoxycarbonyl, and lower alkylcarbonylamino.

The compounds wherein m is an integer of 1-3 (preferably 1) and X is Sor preferably O are of particular interest, when R₁ is phenyl, it ispreferably substituted with 0-3 substituents independently chosen fromhalo, methyl, methoxy, NO₂, trifluoromethyl, and carboxyl. Specificexamples are formed in the Examples.

Another preferred aspect is a compound of formula (I) or formula (II)wherein m is 1 and R₁ is optionally substituted cycloalkyl having 3 to 7carbons forming the ring, optionally substituted fused 2-, 3-, or 4-ringheterocycle, optionally substituted 5- or 6-membered heterocycle, oroptionally substituted anthraquinone.

Another aspect of this invention is a compound of Formula (I) or (II)where R is C(O)—(CH₂)M-X—R′ where m is 0, X is a covalent bond and R′ isa phenyl substituted by a camptothecin or a camptothecin derivativethrough a carbonyl group attached to the E ring of the camptothecin atthe 20 S-oxygen.

This is visualized by referencing U.S. Pat. No. 6,350,756 at columns 7and 8, which results in a compound of formula (I) or formula (II), whereR is C(O)—(CH₂)_(m)—X—R₁, wherein m is 0, X is a covalent bond, and

R₁ is

R₂₂ is hydrogen, halo, lower alkyl, lower alkoxy, hydroxy, R₄₀C(O)O,cyano, nitro, amino, halogenated lower alkyl, halogenated lower alkoxy,hydroxycarbonyl, formyl, lower alkoxycarbonyl, tri lower alkylsilyl,lower alkylcarbonyloxy, lower alkylcarbonylamino, loweralkylcarbonyloxymethyl, substituted vinyl, 1-hydroxy-2-nitroethyl,alkoxycarbonylethyl, aminocarbonyl, mono- or di-alkylcarbonyl,alkylcarbonylmethyl, benzoylmethyl, benzylcarbonyloxymethyl, or mono- ordi lower alkoxymethyl;

R₂₃ is hydrogen, halo, lower alkyl, lower alkoxy, hydroxy, R₄₀C(O)O,cyano, nitro, amino, halogenated lower alkyl, halogenated lower alkoxy,hyroxycarbonyl, formyl, lower alkoxycarbonyl, CH₂NR₂₇R₂₈ (where each ofR₂₇ and R₂₈ is independently H—, alkyl of 1-6 carbons, optionallysubstituted phenyl, hydroxy lower alkyl, amino lower alkyl, or mono- ordialkylamino lower alkyl, or R₂₇ and R₂₈ taken together with —N—represent a cyclic amino-), CH₂R₂₉ (where R₂₉ is lower alkoxy, CN, aminolower alkoxy, mono- or di-lower alkylamino lower alkoxy, loweralkylthio, amino lower alkylthio, or mono- or di-lower alkylamino loweralkylthio), or NR₃₀R₃₁ (where each of R₃₀ and R₃₁ is independentlyhydrogen, lower alkyl, phenyl, hydroxy lower alkyl, amino lower alkyl,or mono- or di-lower alkyl, or R₃₀ and R₃₁ taken together with —N—represent a cyclic amino), dialkylamino alkyl, lower alkylcarbonyloxy,or lower alkylcarbonylamino;

R₂₄ is hydrogen, halo, lower alkyl, lower alkoxy, hydroxy, R₄₀C(O)O,cyano, nitro, amino, amino lower alkyl, halogenated lower alkyl,halogenated lower alkoxy, hydroxycarbonyl, formyl, lower alkoxycarbonyl,carbamoyloxy, lower alkylcarbonyloxy, or lower alkylcarbonylamino, orR₂₄ together with R₂₅ is methylenedioxy;

R₂₅ is hydrogen, halo, lower alkyl, lower alkoxy, hydroxy, R₄₀C(O)O,cyano, nitro, amino, halogenated lower alkyl, halogenated lower alkoxy,hydroxycarbonyl, formyl, lower alkoxycarbonyl, lower alkylcarbonyloxy,or lower alkylcarbonylamino; and

R₂₆ is hydrogen, halo, lower alkyl, lower alkoxy, hydroxy, R₄₀C(O)O,cyano, nitro, amino, halogenated lower alkyl, halogenated lower alkoxy,hydroxycarbonyl, formyl, lower alkoxycarbonyl, lower alkylcarbonyloxy,or lower alkylcarbonylamino;

R₄₀ is R₄₁—O—(CH₂)_(s)—;

s is an integer of 1-10;

R₄₁ is

lower alkyl;

-   -   phenyl optionally substituted with from one to five substituents        independently selected from the group consisting of halo, lower        alkyl, lower alkoxy, hydroxy, cyano, nitro, amino, halogenated        lower alkyl, halogenated lower alkoxy, formyl, lower alkyl        carbonyl, hydroxycarbonyl, lower alkylcarbonyloxy, benzyloxy,        optionally substituted piperazino, lower alkoxycarbonyl, and        lower alkylcarbonylamino;    -   cycloalkyl of 3-7 carbons, optionally substituted with one to        five substituents independently selected from the group        consisting of halo, lower alkyl, lower alkoxy, hydroxy, cyano,        nitro, amino, halogenated lower alkyl, halogenated lower alkoxy,        hydroxycarbonyl, lower alkoxycarbonyl, lower alkylcarbonyloxy,        and lower alkylcarbonylamino;    -   a fused, 2-, 3-, or 4-ring heterocyclic system optionally        substituted with one to five substituents independently selected        from the group consisting of halo, lower alkyl, lower alkoxy,        hydroxy, cyano, nitro, amino, halogenated lower alkyl,        halogenated lower alkoxy, hydroxycarbonyl, lower alkoxycarbonyl,        lower alkylcarbonyloxy, and lower alkylcarbonylamino;    -   1- or 2-naphthyl optionally substituted with from one to four        substituents independently selected from the group consisting of        halo, lower alkyl, lower alkoxy, hydroxy, cyano, nitro, amino,        halogenated lower alkyl, halogenated lower alkoxy,        hydroxycarbonyl, lower alkoxycarbonyl, lower alkylcarbonyloxy,        and lower alkylcarbonylamino; and    -   a 5 or 6 membered heterocyclic ring containing one or two        nitrogen atoms, which ring is optionally substituted with one or        two substituents selected from the group consisting of halo,        lower alkyl, lower alkoxy, hydroxy, cyano, nitro, amino,        halogenated lower alkyl, halogenated lower alkoxy,        hydroxycarbonyl, lower alkoxycarbonyl, lower alkylcarbonyloxy,        and lower alkylcarbonylamino;    -   wherein the wavy line represents the point of connection to X.

Some aspects of the invention include compounds as describedhereinbefore. These include, for example, the preferred subgroups setforth hereinafter:

The compound of formula (I) or (II), wherein R₂₆ is hydrogen,particularly a compound wherein R₂₄ and R₂₅ together are methylenedioxyand wherein R₂₂ is hydrogen. Of these the compounds of particularinterest are those where R₂₃ is nitro, amino, methyl, chloro, cyano,acetoxy, or acetylamino.

A compound of formula (I) or (II), wherein each of R₂₅ and R₂₆ ishydrogen, especially those wherein R₂₃ is hydrogen; R₂₂ is(3-chloro-n-propyl)dimethylsilyl, tert-butyldimethylsilyl,acetoxymethyl, cyano, formylethenyl, ethoxycarbonyl-ethenyl,cyanoethenyl, 2,2-dicyanoethenyl, (2-cyano-2-ethoxycarbony)ethenyl,ethoxycarbonylethyl, methyl, ethyl, or n-propyl; and R₂₄ is hydroxy,acetoxy, amino, nitro, cyano, chloro, bromo, fluoro, lower alkyl, higheralkyl, lower alkoxy, carbamoyloxy, or formyl. Of these, the compoundswherein R₂₂ is ethyl and R₂₄ is carbamoyloxy are of further interest.Carbamoyloxy substituents that are preferred include1-piperazinocarbonyloxy,4-(i-propylaminocarbonylmethyl)piperazin-1-yl-carbonyloxy, or4-(1-piperidino)-1-piperidinocarbonyloxy.

The compound of formula (I) or (II), wherein each of R₂₂, R₂₅, and R₂₆is hydrogen, for example, those wherein R₂₃ is amino, nitro, cyano,halo, OH, lower alkylamino, di-lower alkylamino, lower alkyl, loweralkoxy, 1-piperidino, 1-morpholino, aminomethyl, lower alkylaminomethyl,cycloalkylaminomethyl, di-lower alkylaminomethyl, cyclic aminomethyl,acetoxy, acetylamino, lower alkoxymethyl, omega hydroxy loweralkylaminomethyl cyanomethyl and R₂₄ is hydroxy, acetoxy, cyano, nitro,amino, halo, formyl, lower alkoxy, carbamoyloxy.

A compound wherein each of R₂₂, R₂₃, R₂₅ and R₂₆ is hydrogen and R₂₄ is—OC(O)alkyl₁₋₂₀.

Thus, in one embodiment,

R₂₂ is hydrogen;

R₂₃ is CH₂NR₂₇R₂₈ (where each of R₂₇ and R₂₈ is independently H—, alkylof 1-6 carbons, optionally substituted phenyl, hydroxy lower alkyl,amino lower alkyl, or mono- or dialkylamino lower alkyl, or R₂₇ and R₂₈taken together with —N— represent a cyclic amino-), NR₃₀R₃₁ (where eachof R₃₀ and R₃₁ is independently hydrogen, lower alkyl, phenyl, hydroxylower alkyl, amino lower alkyl, or mono- or di-lower alkyl, or R₃₀ andR₃₁ taken together with —N— represent a cyclic amino), or dialkylaminoalkyl;

R₂₄ is lower alkoxy, hydroxy, halogenated lower alkyl, halogenated loweralkoxy, hydroxycarbonyl, formyl, lower alkoxycarbonyl, carbamoyloxy,lower alkylcarbonyloxy, or R₂₄ together with R₂₅ is methylenedioxy;

R₂₅ is hydrogen, or together with R₂₄ is methylenedioxy; and

R₂₆ is hydrogen.

In another embodiment, where each of R₂₅ and R₂₆ is hydrogen, R₂₃ isCH₂NR₂₇R₂₈ (where each of R₂₇ and R₂₈ is lower alkyl), and R₂₄ ishydroxy, alkoxy or alkylcarbonyloxy. A representative compound is whereR₂₃ is CH₂N(CH₃)₂ and R₂₄ is hydroxy.

In another embodiment,

R₂₂ is hydrogen, lower alkyl, or halogenated lower alkyl;

R₂₃ is hydrogen or lower alkyl;

R₂₄ is lower alkoxy, hydroxy, halogenated lower alkoxy, carbamoyloxy,lower alkylcarbonyloxy, or R₂₄ together with R₂₅ is methylenedioxy;

R₂₅ is hydrogen, or together with R₂₄ is methylenedioxy; and

R₂₆ is hydrogen.

A representative compound is where each of R₂₃, R₂₅ and R₂₆ is hydrogen,R₂₂ is alkyl, such as ethyl, and R₂₄ is carbamoyloxy, such as4-(1-piperazino)-1-piperidino-carbonyloxy.

In yet another embodiment,

R₂₂ is lower alkyl, e.g., ethyl;

each of R₂₃, R₂₅, and R₂₆ is hydrogen; and

R₂₄ is hydroxy, lower alkoxy, halogenated lower alkoxy, hydroxycarbonyl,formyl, lower alkoxycarbonyl, carbamoyloxy, or lower alkylcarbonyloxy,particularly hydroxy.

In yet another embodiment,

each of R₂₂, R₂₄, R₂₅, and R₂₆ is hydrogen; and

R₂₃ is amino or nitro.

In yet another embodiment,

R₂₂ is tri-lower alkylsilyl, such as t-butyldimethylsilyl;

each of R₂₃, R₂₅ and R₂₆ is hydrogen; and

R₂₄ is hydroxy, lower alkoxy, halogenated lower alkoxy, hydroxycarbonyl,formyl, lower alkoxycarbonyl, carbamoyloxy, or lower alkylcarbonyloxy,particularly hydroxy.

The compounds of formula (I) or formula (II) wherein m is an integer of0-3 (preferably 1) and X is oxygen or a covalent bond are of particularinterest. These compounds are of particular interest when X is acovalent bond and R₁ is an optionally substituted 5- or 6-memberedheterocycle with an oxygen or one or two nitrogens in the ring, a fusedheterocyclic ring system, or a fused carbocyclic system. Specificexamples are formed in the Examples.

Pharmaceutical Composition of the Invention

This aspect of the invention is a pharmaceutical composition useful fortreating cancer in a warm-blooded animal, which composition comprisescompound of the invention as defined herein in combination with apharmaceutically acceptable excipient. The composition is prepared inaccordance with known formulation techniques to provide a compositionsuitable for oral, topical, transdermal, rectal, by inhalation,parenteral (intravenous, intramuscular, or intraperitoneal)administration, and the like. Detailed guidance for preparingcompositions of the invention are found by reference to the 18^(th) or19^(th) Edition of Remington's Pharmaceutical. Sciences, Published bythe Mack Publishing Co., Easton, Pa. 18040. The pertinent portions areincorporated herein by reference.

Unit doses or multiple dose forms are contemplated, each offeringadvantages in certain clinical settings. The unit dose would contain apredetermined quantity of active compound calculated to produce thedesired effect(s) in the setting of treating cancer. The multiple doseform may be particularly useful when multiples of single doses, orfractional doses, are required to achieve the desired ends. Either ofthese dosing forms may have specifications that are dictated by ordirectly dependent upon the unique characteristic of the particularcompound, the particular therapeutic effect to be achieved, and anylimitations inherent in the art of preparing the particular compound fortreatment of cancer.

A unit dose will contain a therapeutically effective amount sufficientto treat cancer in a subject and may contain from about 1.0 to 1000 mgof compound, for example about 50 to 500 mg.

The compound will preferably be administered orally in a suitableformulation as an ingestible tablet, a buccal tablet, capsule, caplet,elixir, suspension, syrup, trouche, wafer, lozenge, and the like.Generally, the most straightforward formulation is a tablet or capsule(individually or collectively designated as an “oral dosage unit”).Suitable formulations are prepared in accordance with a standardformulating techniques available that match the characteristics of thecompound to the excipients available for formulating an appropriatecomposition. A tablet or capsule will preferably contain about 50 toabout 500 mg of a compound of Formula (I).

The form may deliver a compound rapidly or may be a sustained-releasepreparation. The compound may be enclosed in a hard or soft capsule, maybe compressed into tablets, or may be incorporated with beverages, foodor otherwise into the diet. The percentage of the final composition andthe preparations may, of course, be varied and may conveniently rangebetween 1 and 90% of the weight of the final form, e.g., tablet. Theamount in such therapeutically useful compositions is such that asuitable dosage will be obtained. Preferred compositions according tothe current invention are prepared so that an oral dosage unit formcontains between about 5.0 to about 50% by weight (% w) in dosage unitsweighing between 5 and 1000 mg.

The suitable formulation of an oral dosage unit may also contain: abinder, such as gum tragacanth, acacia, corn starch, gelatin; sweeteningagents such as lactose or sucrose; disintegrating agents such as cornstarch, alginic acid and the like; a lubricant such as magnesiumstearate; or flavoring such a peppermint, oil of wintergreen or thelike. Various other material may be present as coating or to otherwisemodify the physical form of the oral dosage unit. The oral dosage unitmay be coated with shellac, a sugar or both. Syrup or elixir may containthe compound, sucrose as a sweetening agent, methyl and propylparabensas a preservative, a dye and flavoring. Any material utilized should bepharmaceutically-acceptable and substantially non-toxic. Details of thetypes of excipients useful may be found in the nineteenth edition of“Remington: The Science and Practice of Pharmacy,” Mack PrintingCompany, Easton, Pa. See particularly chapters 91-93 for a fullerdiscussion.

A compound may be administered parenterally, e.g., intravenously,intramuscularly, intravenously, subcutaneously, or intraperitoneally.The carrier or excipient or excipient mixture can be a solvent or adispersive medium containing, for example, various polar or non-polarsolvents, suitable mixtures thereof, or oils. As used herein “carrier”or “excipient” means a pharmaceutically acceptable carrier or excipientand includes any and all solvents, dispersive agents or media,coating(s), antimicrobial agents, iso/hypo/hypertonic agents,absorption-modifying agents, and the like. The use of such substancesand the agents for pharmaceutically active substances is well known inthe art. Except insofar as any conventional media or agent isincompatible with the active ingredient, use in therapeutic compositionsis contemplated. Moreover, other or supplementary active ingredients canalso be incorporated into the final composition.

Solutions of the compound may be prepared in suitable diluents such aswater, ethanol, glycerol, liquid polyethylene glycol(s), various oils,and/or mixtures thereof, and others known to those skilled in the art.

The pharmaceutical forms suitable for injectable use include sterilesolutions, dispersions, emulsions, and sterile powders. The final formmust be stable under conditions of manufacture and storage. Furthermore,the final pharmaceutical form must be protected against contaminationand must, therefore, be able to inhibit the growth of microorganismssuch as bacteria or fungi. A single intravenous or intraperitoneal dosecan be administered. Alternatively, a slow long term infusion ormultiple short term daily infusions may be utilized, typically lastingfrom 1 to 8 days. Alternate day or dosing once every several days mayalso be utilized.

Sterile, injectable solutions are prepared by incorporating a compoundin the required amount into one or more appropriate solvents to whichother ingredients, listed above or known to those skilled in the art,may be added as required. Sterile injectable solutions are prepared byincorporating the compound in the required amount in the appropriatesolvent with various other ingredients as required. Sterilizingprocedures, such as filtration, then follow. Typically, dispersions aremade by incorporating the compound into a sterile vehicle which alsocontains the dispersion medium and the required other ingredients asindicated above. In the case of a sterile powder, the preferred methodsinclude vacuum drying or freeze drying to which any required ingredientsare added.

In all cases the final form, as noted, must be sterile and must also beable to pass readily through an injection device such as a hollowneedle. The proper viscosity may be achieved and maintained by theproper choice of solvents or excipients. Moreover, the use of molecularor particulate coatings such as lecithin, the proper selection ofparticle size in dispersions, or the use of materials with surfactantproperties may be utilized.

Prevention or inhibition of growth of microorganisms may be achievedthrough the addition of one or more antimicrobial agents such aschlorobutanol, ascorbic acid, parabens, thermerosal, or the like. It mayalso be preferable to include agents that alter the tonicity such assugars or salts.

Although the compounds of this invention tend to be water soluble, insome cases, e.g., where a compound of the invention is less watersoluble, it may be useful to provide liposomal delivery. The systemrestrains the compound of the invention by incorporating, encapsulating,surrounding, or entrapping the compound of the invention in, on, or bylipid vesicles or liposomes, or by micelles.

Liposomes have been used successfully to administer medications tocancer patients, and have been shown to be useful clinically in thedelivery of anticancer drugs such as doxorubicin, daunorubicin, andcisplatinum complexes. Forssen, et al., Cancer Res. 1992, 52: 3255-3261;Perex-Soler, et al., Cancer Res. 1990, 50: 4260-4266; and, Khokhar, etal., J. Med. Chem. 1991, 34: 325-329, all of which are incorporatedherein in their entireties by reference.

Similarly, micelles have also been used to deliver medications topatients, (Broden et al., Acta Pharm Suec. 19: 267-284 (1982)) andmicelles have been used as drug carriers and for targeted drug delivery,(D. D. Lasic, Nature 335: 279-280 (1992); and, Supersaxo et al., PharmRes. 8: 1280-1291 (1991)), including cancer medications, (Fung et al.,Biomater. Artif. Cells. Artif. Organs 16: 439 et seq. (1988); andYokoyama et al., Cancer Res. 51: 3229-3236 (1991)), al of which areincorporated herein in their entireties by reference.

The liposomes and/or micelles containing the compound of the inventioncan be administered to a cancer patient, typically intravenously.Further guidance for preparing liposomal compositions useful in thisinvention may be found in U.S. Pat. No. 6,096,336, which is incorporatedherein by reference.

Method of Treatment of the Invention

Another aspect of this invention is a method for treating cancer in awarm-blooded animal, which method comprises administering atherapeutically effective amount of a compound of the invention asdefined herein. A compound useful in this invention is administered toan appropriate subject in need of these compounds in a therapeuticallyeffective dose by a medically acceptable route of administration such asorally, parentally (e.g., intramuscularly, intravenously,subcutaneously, interperitoneally), transdermally, rectally, byinhalation and the like.

The term cancer is to be considered in the broadest general definitionas a malignant neoplasm, an abnormal mass of tissue, the growth of whichexceeds and is uncoordinated with that of normal tissues and persists inthe same excessive manner after cessation of the stimuli that evoked thechange. It might be added that the abnormal mass is purposeless, preyson the host, and is virtually autonomous. A cancer can also beconsidered as a malignant tumor. A further discussion of neoplasia isfound at “Robbins Pathologic Basis of Disease,” Sixth Edition, by R. S.Cotran, V. Kumar, and T. Collins, Chapter 8 (W.B. Saunders Company).This information from Chapter 8 is incorporated herein by reference. Thefollowing Table A provides examples of the types of cancers, i.e.,malignant tumors or neoplasia that may be treated by administering acompound of this invention. TABLE A Tissue of Origin Malignant Composedof One Parenchymal Cell Type Mesenchymal tumors Connective tissue andderivatives Fibrosarcoma Liposarcoma Chondrosarcome Osteogenic sarcomaEndothelial and related tissues Blood vessels Angiosarcoma Lymph vesselsLymphangiosarcoma Synovium Synovial sarcoma Mesothelium MesotheliomaBrain coverings Invasive meningioma Blood cells and related cellsHematopoietic cells Leukemias Lymphoid tissue Malignant lymphomas MuscleSmooth Leiomyosarcoma Straited Rhabdomyosarcoma Epthelial tumorsStratified squamous Squamous cell or epidermoid carcinoma Basal cells ofskin or adnexa Basal cell carcinoma Epithelial lining Glands or ductsAdenocarcinoma Papillary carcinoma Cystadenocarcinoma Respiratorypassages Bronchogenic carcinoma Bronchial adenoma (carcinoid)Neuroectoderm Malignant melanoma Renal epithelium Renal cell carcinomaLiver cells Hepatocellular carcinoma Urinary tract epitheliumTransitional cell carcinoma (transitional) Placental epithelium(trophoblast) Choriocarcinoma Testicular epithelium (germ cells)Seminoma Embryonal carcinoma More Than One Neoplastic Cell-Mixed Tumors,Usually Derived From One Germ Layer Salivary glands Malignant mixedtumor of salivary gland origin Breast Malignant cystosarcoma phyllodesRenal anlage Wilms tumor More Than One Neoplastic Cell Type Derived FromMore Than One Germ Layer-Teratogenous Totipotential cells in gonads orImmature teratoma, teratocarcinoma in embryonic rests

The compounds of the invention are thus useful in the treatment ofleukemia and solid tumors, such as colon, colo-rectal, ovarian, mammary,prostate, lung, kidney and also melanoma tumors. The dosage rangeadopted will depend on the route of administration and on the age,weight and condition of the patient being treated. The compounds may beadministered, for example, by the parenteral route, for example,intramuscularly, intravenously or by bolus infusion.

As used herein, a “therapeutically effective amount” of podophyllotoxinderivatives of the present invention is intended to mean that amount ofthe compound which will inhibit the growth of, or retard cancer, or killmalignant cells, and cause the regression and palliation of malignanttumors, i.e., reduce the volume or size of such tumors or eliminate thetumor entirely.

With mammals, including humans, the effective amounts can beadministered on the basis of body surface area. The interrelationship ofdosages varies for animals of various sizes and species, and for humans(based on mg/m² of body surface) is described by E. J. Freireich et al.,Cancer Chemother. Rep., 50(4):219 (1966). Body surface area may beapproximately determined from the height and weight of an individual(see, e.g., Scientific Tables, Geigy Pharmaceuticals, Ardsley, N.Y. pp.537-538 (1970)). A suitable dose range is from 1 to 1000 mg ofequivalent per m² body surface area of a compound of the invention, forinstance from 50 to 500 mg/m².

For all of the administering routes, the exact timing of administrationof the dosages can be varied to achieve optimal results. Generally, ifusing Intralipid 20 as the carrier for the derivative, the actual dosageof derivative reaching the patient will be less. This is due to someloss of the derivative on the walls of the syringes, needles andpreparation vessels, which is prevalent with the Intralipid 20suspension. When a carrier, such as cottonseed oil is used, this abovedescribed loss is not so prevalent because the derivative does notadhere as much to the surface of syringes, etc.

Another important feature of the method provided by the presentinvention relates to the relatively low apparent overall toxicity of thederivatives administered in accordance with the teachings herein.Overall toxicity can be judged using various criteria. For example, lossof body weight in a subject over 10% of the initially recorded bodyweight (i.e., before treatment) can be considered as one sign oftoxicity. In addition, loss of overall mobility and activity and signsof diarrhea or cystitis in a subject can also be interpreted as evidenceof toxicity.

Process of the Invention

Another aspect of this invention is process for preparing compounds ofthis invention by reacting podophyllotoxin (PT) or4′-demethylepipodophyllotoxin (DPT) with a compound of the formulaYC(O)(CH₂)_(m)XR₁, wherein m, X, and R₁, are as defined herein, and Y ise.g. bromide, chloride, hydroxy, or alkoxy. Preferably Y is OH. Thecompound shown as YC(O)(CH₂)_(m)XR₁, can be referred to as a substitutedalkanoic acid or substituted alkanoic acid derivative, e.g. where m is1, it is substituted acetic acid or a derivative thereof, where m is 2,it is a substituted propionic acid or a derivative thereof, etc. One waythat such an alkanoic acid is obtained is by reacting an appropriateR₁XH compound with an omega-halosubstituted alkanoic acid ester (e.g.3-halopropionic ester or 3-haloacetic ester), then hydrolyzing the esterto form the acid. Examples of preferred haloacetic acid or halopropionicacid esters include the ethyl ester of 2 or 3-bromo acid, 3-chloro acid,or 2 or 3-iodo acid. Other corresponding alkyl esters (e.g., methyl,propyl, and the like, are useful but ethyl is preferred). In some cases,it may be useful to prepare an acid halide from the correspondingalkanoic acid. The acid halides are obtained by reacting thecorresponding acid with halogenated agents (such as SOCl₂, PCl₃, POCl₃,PCl₅, PBr₃, and so on). The acid chloride is preferred. Once the acid orits derivative is prepared, it is reacted with podophyllotoxin or4′-demethylepipodophyllotoxin to form a compound of this invention. Thisreaction sequence can be generalized as follows:

In the reaction sequence PT represents podophyllotoxin or4′-demethylepipodophyllotoxin; Y is hydroxy, halo, or alkoxy; m is aninteger of 0-10; X is oxygen, sulfur, nitrogen, or a covalent bond; andR₁ is as defined herein.

In the reaction sequence above, compound (A) will be used in molarexcess of compound (B), e.g. a molar ratio of about 1.5:1 to about 4:1,preferably about 2:1 to 3:1. The reaction takes place in the presence ofsuitable coupling agent such as a carbodiimide compound, e.g.disopropylcarbodiimide, but preferably 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) and 4-(dimethylamino) pyridine (DMAP)in the presence of a suitable solvent, preferably a nonaqueous, nonpolarsolvent. Examples of useful solvents in this step include halogenatedalkane (e.g., dichoromethane or trichloromethane) and DMF.Dichloromethane is particularly useful. The reaction temperature willrange from about 20° C. to about 40° C., preferably about 20° C. toabout 25° C. The time needed for the reaction to be complete willgenerally be no more than about 20 hours, usually less than about 10hours.

The podophyllotoxin or 4′-demethylepipodophyllotoxin compounds areavailable to those of skill in the art by purchasing from Sigma-Aldrich,St. Louis, Mo. (podophyllotoxin) or Medichem China Group Company, HongKong (4′-demethylepipodophyllotoxin).

The compound of formula (II) may be converted to the corresponding4′-phosphate ester as follows: A suspension of 50% NaH in mineral oiland the 4′-demethylepipodophyllotoxin-4-O-ester of formula (II) of isformed in an organic solvent. The mixture is cooled to 0° C.; a solutionof dibenzylphosphorochloridate in toluene is added drop by drop. Afterthe reaction comes to completion, the solution is diluted with coldwater and extracted with ether. The ether solution is washed with water,dried, evaporated under reduced pressure to give the4′-dibenzylphosphate derivative of the compound of formula (II). Asolution of the dibenzylphosphate derivative in 85% ethanol ishydrogenated in a Parr apparatus in the presence of 10% Pd supported oncarbon. After theoretical absorption of hydrogen, the catalyst isfiltered, washed with cold water and suspended in 2N NH₄OH at 50° C. Thesuspension is filtered, washed with water and concentrated at reducedpressure at 50° C. The solution is then filtered and acidified with HClto give a 4′-phosphate ester of a compound of formula (II).

It will be recognized by one of skill in the art that other similarcompounds may be prepared by following the teachings set forth in theabove articles and modifying with appropriate art-recognized steps.

Suitable alkanoic acids of formula (A) are available commercially andinclude the following (see the catalog by the Sigma-Aldrich Corp., St.Louis, Mo. or www.sigmaaldrich.com):

-   4-fluorophenoxyacctic acid;-   2,4-dimethylphenoxyacetic acid;-   4-methoxyphenoxyacetic acid;-   4-formylphenoxyacetic acid;-   2-nitrophenoxyacetic acid;-   5-nitro-2-furoic acid;-   3-chloroacetaminobenzoic acid;-   (4-pyridylthio) acetic acid;-   chromone-2-carboxylic acid;-   anthraquinone-2-carbonyl chloride;-   1H-tetrazole-1-acetic acid;-   (4-chlorophenylthio) acetic acid;-   quinoline-4-oxyacetic acid;-   4-nitrophenoxyacetic acid;-   3,5-ditrifluoromethylphenoxyacetic acid;-   4-trifluoromethoxyphenenoxyacetic acid;-   4-bromophenoxyacetic acid;-   4-iodophenoxyacetic acid; phenoxyacetic acid;-   2,4-dichloro-5-methylphenylthioacetic acid;-   2,3,4,5,6-pentafluorophenoxyacetic acid;-   3-fluoro-4-cyanophenoxyacetic acid;-   3-trifluoromethyl-4-nitrophenoxyacetic acid;-   4-phthalimidobenzoic acid;-   3-chloro-4-bromophenoxy acetic acid;-   2,6-diiodo-4-cyanophenoxyacetic acid;-   4-(2-phenyl) quinolinecarboxylic acid;-   phenothiazin-10-ylcarbonylchloride;-   1,7-dimethyl-naphthyridin-4-one-3-carboxylic acid;-   3-pyridinepropionic acid;-   4-chlorophenoxyacetic acid;-   3-methoxyphenoxyacetic acid;-   thymine-1-acetic acid;-   (+)-2-(2,4,5,7-tetranitro-9-fluorenylideneaminooxy) propionic acid;-   3-phthalimidopropionic acid;-   3-maleimidopropionic acid;-   3-(3-nitro 1,8-naphthalimide)propionic acid;-   3-(4-nitro-1,8-naphthalimide)propionic acid;-   3-(4-bromo-1,8-naphthalimido)propionic acid;-   3-[4-(3-trifluoromethylphenyl)-1-piperazinyl]-propionic acid;-   3-[(4-benzyl)-1-piperazinyl]propionic acid;-   3-[4-(3-methoxyphenyl)-1-piperazinyl]propionic acid;-   3-[4-(4-nitrophenyl)-1-piperazinyl]propionic acid;-   3-(4-phenyl-1-piperazinyl)propionic acid;-   3-[4-(2-chlorophenyl)-1-piperazinyl]propionic acid;-   3-[4-(4-fluorophenyl)-1-piperazinyl]propionic acid;-   3-(1-piperidino)propionic acid;-   3-[1-(4-benzyl)piperidino]propionic acid;-   3-[4-(4-acetylphenyl-1-piperazinyl]propionic acid;-   3-[4-(3,4-dichlorophenyl)-1-piperazinyl]propionic acid;-   3-[4-(3,4-methylenedioxyphenyl)-1-piperazinyl]propionic acid;-   3-[4-(4-chlorophenyl)-1-piperidinyl]propionic acid;-   3-(4-formyl-1-piperazinyl)propionic acid;-   3-(4-ethyl-1-piperazinyl)propionic acid;-   3-[4-(4-chlorophenyl)phenylmethyl-1-piperazinyl]propionic acid;-   3-(4-cyano-4-phenyl-1-piperidinyl) propionic acid;-   3-trans-4-cinnamyl-1-piperazinyl) propionic acid;-   3-[4-(2-methylphenyl)-1-piperazinyl]propionic acid;-   3-[4-(2,3-dimethylphenyl)-1-piperazinyl]propionic acid;-   3-[4-(1-piperidino)-1-piperidino]propionic acid;-   3-[4-(2-pyrimidinyl)-1-piperazinyl]propionic acid;-   3-(4-cyclohexyl-1-piperazinyl) propionic acid;-   3-[4-(α-(2-pyridyl)benzyl-1-piperazinyl]propionic acid;-   3-(4-morpholino)propionic acid;-   3-(1-pyrrolinyl)propionic acid;-   4-[4-(3-trifluoromethylphenyl)-1-piperazinyl]butyric acid;-   5-[4-(3-trifluoromethylphenyl)-1-piperazinly]valeric acid; and the    like.

One of skill in the art will recognize that other similar 3-propionicacids and 2-acetic acids may be obtained from commercial sources orprepared by art-recognized procedures to be used in the process toprepare compounds of this invention. By reacting podophyllotoxin or4′-demethylepipodophyllotoxin with a compound shown in the list ofcompounds of formula (A) in accordance with the guidelines for reactioncondition, compounds of the invention will be obtained. These compoundswill exhibit the desired characteristics to a greater or lesser extent.Guidance is provided herein as to the preferred subgroups of compoundswithin the family.

Another aspect of this invention is process for preparing compounds ofthis invention by following the teachings of U.S. Pat. No. 6,350,756 atcolumns 16, line 46, to column 18, line 19. The process is carried outby reacting podophyllotoxin (PT) or 4′-demethylepipodophyllotoxin (DPT)with a compound of the formula Y′C(O)—(CH₂)_(m)R₂₀—C(O)—CPT, wherein mis 0, R₂₀ is phenyoxymethyl, Y′ is e.g., bromide, chloride, hydroxyl, oralkoxy, and CPT in this formula represents both camptothecin andcamptothecin derivatives. Preferably, Y′ is hydroxyl. When Y′ is alkoxy,Y′C(O)(CH₂)_(m)R₂₀—C(O)O—CPT may be prepared by reacting camptothecin(CPT) or a CPT analog with a compound of the formulaY′C(O)(CH₂)_(m)R₂₀—C(O)X₂, wherein X₂ is e.g. bromide, chloride, orhydroxy. Preferably X₂ is OH. One way to prepare the compound shown asY′C(O)—(CH₂)_(m)R₂₀—C(O)X₂ is by reacting an appropriate alcohol (Y′OH)with HalC(O)—(CH₂)_(m)R₂₀—C(O)X₂ (where X₂ is alkoxy), then hydrolyzing.HalC(O)—(CH₂)_(m)R₂₀—C(O)X₂ (where X₂ is alkoxy) may be prepared fromthe corresponding acid HO—C(O)—(CH₂)_(m)R₂₀—C(O)X₂ (where X₂ is alkoxy)via a reaction with halogenated agents (such as SOCl₂, PCl₃, POCl₃,PCl₅, PBr₃, and so on). The acid chloride is preferred. OnceY′C(O)(CH₂)_(m)R₂₀—C(O)X₂ is prepared, it is reacted with CPT or a CPTanalog to form the (S)-20-ester of CPT, i.e.Y′C(O)(CH₂)_(m)R₂₀—C(O)O—CPT, which can then be used to prepare thecorresponding PT or DPT esters of this invention according to theprocedure described above. This reaction sequence can be generalized asfollows:

In step 1 the reaction conditions will vary depending on the exactreactants employed. In general, solvents useful in the reaction may beaqueous or nonaqueous. Preferably, a solvent will be water, an organicsolvent miscible with water, or mixtures thereof. Examples of usefulmiscible solvents include acetone and dimethylormamide (DMF). When thesolvent is aqueous, the pH of the reaction will be basic, e.g. in therange of 10 to 14, preferably about 12 to 14. The reaction temperaturevaries with the reactants, and the solvents, and will range from about20° C. to about 180° C., preferably about 40° C. to about 80° C. Thetime needed for the reaction to be complete will generally be no morethan about 10 hours, preferably about 2 to 4 hours.

In step 2, the compound of formula (C′) is converted to a compound offormula (D′) by a hydrolysis reaction, generally performed in twostages. The reaction conditions for this step will vary in accordancewith the compound being reacted. In general, solvents useful in theconversion may be aqueous or nonaqueous, preferably, a solvent will bewater, either alone or with a water-miscible organic solvent. An exampleof a particularly useful solvent is a mixture of water and DMF or waterand dioxane. The pH of the first stage of reaction will be basic, e.g.in the range of 10 to 14, preferably about 12 to 14. A suitableinorganic base such as an alkaline earth hydroxide, e.g. sodiumhydroxide, is useful. The reaction temperature will range from about 0°C. to about 60° C., preferably about 20° C. to about 25° C. The timeneeded for the reaction to be complete will generally be no more than 10hours, preferably no more than about 4 hours. The mixture is thenacidified to a pH of less than 4, e.g. 3, with an appropriate acid suchas hydrogen chloride and extracted with a suitable solvent such as ethylacetate in accordance with standard chemical synthetic methods. Reactionselectivity between the two esters Y′C(O)— and —C(O)X₂ can be achievedby using different types of groups. For example, Y′C(O)— may beC(CH₃)₃C(O)—, which is stable to hydrolysis under basic conditions,while —C(O)X₂ can be —C(O)OCH₃, which is liable to base hydrolysis. Suchesters that can be selectively hydrolyzed are known to those skilled inthe art.

In step 2′, the compound of formula D′ (i.e. the oxyalkanoic acid isconverted into the corresponding acid halide D″ by reacting with ahalogenated agent such as SOCl₂, PCl₃, POCl₃, PCl₅, and PBr₃, and thelike under appropriate conditions.

In step 3 of the process, a compound of formula (D′) or (D″) is reactedwith CPT in about equimolar amounts under conditions suitable for theformation of the compounds of this invention as the 20-(S) stereoisomerY′C(O)(CH₂)_(m)R₂₀—C(O)O—CPT. The reaction takes place in the presenceof suitable carbodiimide compound such as diisopropylcarbodiimide, butpreferably 1-(3-dimethylaminopropyl)-3-ethyl carbodiimide hydrochloride(EDCI), and 4-(dimethylamino) pyridine (DMAP) in the presence of asuitable solvent, preferably a nonaqueous, nonpolar solvent. Examples ofuseful solvents in this step include halogenated alkanes, e.g.,dichoromethane or trichloromethane) and DMF. Dichloromethane isparticularly useful. The reaction temperature will range from about 20°C. to about 40° C., preferably about 20° C. to about 25° C. The timeneeded for the reaction to be complete will generally be no more thanabout 20 hours, preferably about 10 hours.

In step 3, a suitable Camptothecin derivative or analog is a compoundthat is camptothecin substituted at the 7, 9, 10, 11, or 12 positions asdescribed in this document. The camptothecin analog may be substitutedwith substituents known in the art or that can be prepared by one ofskill in the art given the disclosure herein. Representative articlesthat teach how to make such analogs or where such analogs may beprocured are found in the following journals (which are incorporatedherein by reference): 1. J. Med. Chem. 1998, 41, 31-37 2. J. Med. Chem.2000, 43, 3970-3980 3. J. Med. Chem. 1993, 36, 2689-2700 4. J. Med.Chem. 1991, 34, 98-107 5. J. Med. Chem. 2000, 43, 3963-3969 6. Chem.Pharm. Bull. 39(10) 2574-2580 (1991) 7. Chem. Pharm. Bull. 39(6)1446-1454 (1991) 8. ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, December1999, p. 2862-2868 9. European Journal of Cancer, Vol. 34, No. 10, pp.1500-1503, 1998 10. CANCER RESEARCH 55, 753-760, Feb. 15, 1995 11.Anti-Cancer Drug Design (1998), 13, 145-157 12. Bioorganic & MedicinalChemistry Letters 8 (1998) 415-418.

Suitable camptothecin analogs include the following, where the number inparenthesis following the name refers to journal article listed above:

-   camptothecin (CPT);-   (20S)-7-ethyl-10-[4-(1-piperidino)-1-piperidino]carbonyloxy)-CPT    (AKA-irinotecan);-   (20S)-9-nitro CPT (1);-   (20S)-7-chloro-n-propyldimethylsilyl CPT (2);-   (20S)-10-hydroxy-7-chloro-n-propyldimethylsilyl CPT (2);-   (20S)-10-acetoxy-7-chloro-n-propyldimethylsilyl CPT (2);-   (20S)-7-tert-butyldimethylsilyl CPT (2);-   (20S)-10-hydroxy-7-tert-butyldimethylsilyl CPT (2);-   (20S)-10-acetoxy-7-tert-butyldimethylsilyl CPT (2);-   (20S)-9-hydroxy CPT (3);-   (20S)-9-amino CPT (3);-   (20S)-10-amino CPT (3);-   (20S)-9-amino-10-hydroxy CPT (3);-   (20S)-9-amino-10,11-methylenedioxy CPT (3);-   (20S)-9-methylamino CPT;-   (20S)-9-methyl CPT (3);-   (20S)-9-dimethylanomethyl CPT;-   (20S)-9-chloro CPT (3);-   (20S)-9-fluoro CPT (3);-   (20S)-9-piperidino CPT;-   (20S)-9-dimethylaminomethyl-10-hydroxy CPT (3)-AKA topotecan);-   (20S)-9-morpholinomethyl CPT (4);-   (20S)-10-hydroxy CPT (3);-   (20S)-9,10-dichloro CPT (3);-   (20S)-10-bromo CPT (3);-   (20S)-10-chloro CPT (3);-   (20S)-10-methyl CPT (3);-   (20S)-10-fluoro CPT (3);-   (20S)-10-nitro CPT (3);-   (20S)-10,11-methylenedioxy CPT (3);-   (20S)-10-formyl CPT (3);-   (20S)-10-nonylcarbonyloxy CPT (12);-   (20S)-10-undecylcarbonyloxy CPT (12);-   (20S)-10-pentadecylcarbonyloxy CPT (12);-   (20S)-10-heptadecylcarbonyloxy CPT (12);-   (20S)-10-nonadecylcarbonyloxy CPT (12);-   (20S)-9-nitro-10,11-methylenedioxy CPT (3);-   (20S)-9-(4-methylpiperazinylmethyl)-10-hydroxy (CPT) (4);-   (20S)-9-[4-(1-piperidino)-1-piperidinomethyl]-10-hydroxy CPT (4);-   (20S)-9-methyl-10,11-methylenedioxy CPT;-   (20S)-9-chloro-10,11-methylenedioxy CPT (3);-   (20S)-9-cyano-10,11-methylenedioxy CPT;-   (20S)-9-acetoxy-10,11-methylenedioxy CPT;-   (20S)-9-acetylamino-10,11-methylenedioxy CPT;-   (20S)-9-aminomethyl-10-hydroxy CPT;-   (20S)-9-ethoxymethyl-10-hydroxy CPT (4);-   (20S)-9-methylaminomethyl-10-hydroxy CPT;-   (20S)-9-n-propylaminomethyl-10-hydroxy CPT (4);-   (20S)-9-dimethylaminomethyl-10-hydroxy CPT (4);-   (20S)-9-cyclohexylaminomethyl-10-hydroxy CPT (4);-   (20S)-9-(2-hydroxyethyl)aminomethyl-10-hydroxy CPT (4);-   (20S)-9-(trimethylammonio)methyl-10-hydroxy CPT, methanesulfonate    (4);-   (20S)-9-morpholinomethyl-10-hydroxy CPT (4);-   (20S)-9-cyanomethyl-10-hydroxy CPT (4); (20S)-CPT-7-aldehyde (5);-   (20S)-10-methoxy CPT-7-aldehyde (5);-   (20S)-7-acetoxymethyl CPT (5);-   (20S)-7-acetoxymethyl-10-methyl CPT (5);-   (20S)-7-cyano-10-methoxy CPT (5);-   (20S)-7-cyano CPT (5);-   (20S)-7-formylethenyl CPT (5);-   (20S)-7-ethoxycarbonylethenyl CPT (5);-   (20S)-7-cyanoethenyl CPT (5);-   (20S)-7-(2,2-dicyanoethenyl) CPT (5);-   (20S)-7-(2-cyano-2-ethoxycarbonyl)ethenyl CPT (5);-   (20S)-7-ethoxycarbonylethyl CPT (5);-   (20S)-7-ethyl CPT (6);-   (20S)-7-n-propyl CPT (6);-   (20S)-7-acetoxymethyl CPT (6);-   (20S)-7-n-propylcarbonyloxymethyl CPT (6);-   (20S)-7-ethoxycarbonyl CPT (6);-   (20S)-7-ethyl-10-hydroxy CPT;-   (20S)-7-ethyl-10-acetyloxy CPT;-   (20S)-7-methyl-10-aminocarbonyloxy CPT;-   (20S)-7-n-propyl-10-piperidinocazbonyloxy CPT;-   (20S)-7-ethyl-10-(2-dimethylamino)ethyl CPT; and-   (20S)-7-ethyl-10-carbamoyloxy derivatives of CPT such as-   (20S)-7-ethyl-10-[4(1-piperidino)-piperidino carbonyloxy CPT (7);-   (20S)-7-ethyl-10-(1-piperazine)carbonyloxy CPT (7);-   (20S)-7-ethyl-10-(4-1-propylaminocarbonylmethylpiperazine)carbonyloxy    CPT (7);-   (20S)-7-ethyl-10-[4(1-pyrrolidinyl)piperazine]carbonyloxy CPT (7);-   (20S)-7-ethyl-10-[(4-dimethylamino)-1-piperidino]carbonyloxy CPT    (7);-   (20S)-7-ethyl-10-[4-(di-n-propylamino)-1-piperidinol]carbonyloxy CPT    (7);-   (20S)-7-ethyl-10-[(4-(di-n-butylamino)-1-piperidino]carbonyloxy CPT    (7);-   (20S)-7-ethyl-10-[4-(1-pyrrolidino)-1-piperidino)]carbonyloxy CPT    (7);-   (20S)-7-ethyl-10-[4-(1-piperidino)-1-piperidino]carbonyloxy CPT (7);-   (20S)-7-ethyl-10-[N-methyl-N-2-(dimethylamino)ethylamino]carbonyloxy    CPT (7) and the like.

In step 4, Y′C(O)(CH₂)_(m)R₂₀—C(O)O—CPT is hydrolyzed toHOC(O)(CH₂)_(m)R₂₀—C(O)O—CPT. In the case where Y′ is C(CH₃)₃—O, thehydrolysis can be performed under acidic conditions using an organicsolvent, such as using hydrochloric acid in methanol or trifluoroaceticacid in dichloromethane.

In step 5, HOC(O)(CH₂)_(m)R₂₀—C(O)O—CPT is reacted with PT or a PTderivative to form a compound of this invention according to theprocedures described above.

Alternatively both PT or a PT derivative and a CPT or CPT derivative canreact with 4-carboxylicphenoxyacetic acid in a one-pot reaction to forma mixture of products (such as PT-CO-Ph-O(CH₂)—C(O)—CPT,CPT-CO-Ph-O(CH₂)—C(O)—PT, PT-CO-Ph-O(CH₂)—C(O)—PT,CPT-CO-Ph-O(CH₂)—C(O)—CPT), which can be separated by conventionalseparation techniques.

EXAMPLES

The following examples are given to provide representative compoundsincluded as part of this invention. The examples also providedescriptions of in vitro and in vivo assays to aid in determining theutility of the compounds. The compounds in examples 1-26 were preparedby reacting an appropriate acid with podophyllotoxin or the 4-demethylepimer. Throughout the examples chemical formulas will be used to namecompounds (e.g. NaHCO₃ is sodium bicarbonate) as appropriate.

Example 1 A. Podophyllotoxin-4-O-ester of 4-fluorophenoxyacetic acid(000615)

The mixture of podophyllotoxin (20 mg, 0.048 mmol),4-fluorophenoxyacetic acid (17 mg, 0.1 mmol), EDCI (40 mg, 0.21 mmol),DMAP (2 mg, 0.02 mmol) and dichloromethane (3 ml) were stirred in theroom temperature for 20 h, then dichloromethane (20 ml) was added to thesolution. Organic layer was washed with water (20 ml), saturated NaHCO₃aqueous solution (10 ml) and brine (20 ml), and then dried over MgSO₄.After the solvent was removed under reduced pressure, the resultingliquid was separated by column chromatography (eluent: ethyl acetate andpetroleum ether) to afford 12 mgpodophyllotoxin-4-O-4-fluorophenoxyacetate, mp.

The chemical structure analysis was performed by ¹HNMR (CDCl₃, 600 MHz);δ 7.01 (t, 2H, Ar—H), 6.88 (q, 2H, Ar—H), 6.62 (s, 1H, Ar—H), 6.54 (s,1H, Ar—H), 6.37 (s, 2H, Ar—H), 5.99 (d, 3H, OCH₂O), 4.73 (q, 2H,COCH₂O), 4.60 (d, 1H, H4), 4.34 (t, 1H, H11), 4.20 (t, 1H, H11), 3.81(s, 3H, OCH₃), 3.74 (s, 6H, OCH₃), 2.95 (d, 1H, H2), 2.85 (m, 1H, H3).

B. 4′-demethylepipodophyllotoxin-4-O-ester of 4-fluorophenoxyacetic acid

By following the procedure of Part A of this Example, but substituting4′-demethylepipodophyllotoxin for podophyllotoxin, one obtains thecorresponding 4′-demethylepipodophyllotoxin compound.

C. 4′-Phosphate ester of 4′-demethylepipodophyllotoxin-4-O-ester of4-fluorophenoxyacetic acid

The compound prepared in part B of this Example is converted to thecorresponding 4′-phosphate ester as follows: A suspension of 50% NaH inmineral oil and the 4′-demethylepipodophyllotoxin-4-O-ester of Part B ofthis Example in DMF is stirred for 30 minutes at room temperature. Afterthe mixture is cooled to 0° C., a solution ofdibenzylphosphorochloridate in toluene is added drop by drop. Thesolution is stirred at room temperature for 15 minutes and then dilutedwith cold water and extracted with ether. The ether solution is washedwith water, dried, evaporated under reduced pressure to give the4′-dibenzylphosphate derivative of the compound of part B. A solution ofthe dibenzylphosphate derivative in 85% ethanol is hydrogenated in aParr apparatus in the presence of 10% Pd supported on carbon. Aftertheoretical absorption of hydrogen, the catalyst is filtered, washedwith cold water and suspended in 2N NH₄OH at 50° C. The suspension isfiltered, washed with water and concentrated at reduced pressure at 50°C. The solution is then filtered and acidified with HCl to give thetitle compound.

Example 2 A. Podophyllotoxin-4-O-ester of 4-bromophenoxyacetic acid(000320)

The mixture of podophyllotoxin (20 mg, 0.048 mmol), 4-bromophenoxyaceticacid (22 mg, 0.1 mmol), EDCI (25 mg, 0.13 mmol), DMAP (2 mg, 0.02 mmol)and dichloromethane (3 ml) were stirred in the room temperature for 20h, then dichloromethane (20 ml) was added to the solution. Organic layerwas washed with water (20 ml), saturated NaHCO₃ aqueous solution (10 ml)and brine (20 ml), and then dried over MgSO₄. After the solvent wasremoved under reduced pressure, the resulting liquid was separated bycolumn chromatography (eluent: ethyl acetate and petroleum ether) toafford 21 mg podophyllotoxin-4-O-4-bromophenoxyacetate, mp.

The chemical structure analysis was performed by ¹HNMR (CDCl₃, 600 MHz);δ 7.42 (t, 2H, Ar—H), 6.81 (d, 2H, Ar—H), 6.62 (s, 1H, Ar—H), 6.54 (s,1H, Ar—H), 6.37 (s, 2H, Ar—H), 5.99 (d, 3H, OCH₂O), 4.74 (q, 2H, COCH₂O,4.60 (d, I H, H4), 4.37 (t, 1H, H11), 4.21 (t, 1H, H11), 3.81 (s, 3H,OCH₃), 3.74 (s, 6H, OCH₃), 2.95 (d, 1H, H2), 2.85 (m, 1H, H3).

B. 4′-demethylepipodophyllotoxin-4-O-ester of 4-bromophenoxyacetic acid

By following the procedure of Part A of this Example, but substituting4′-demethylepipodophyllotoxin for podophyllotoxin, one obtains thecorresponding 4′-demethylepipodophyllotoxin compound.

C. 4′-Phosphate ester of 4′-demethylepipodophyllotoxin-4-O-ester of4-bromophenoxyacetic acid

The compound prepared in part B of this Example is converted to thecorresponding 4′-phosphate ester as follows: A suspension of 50% NaH inmineral oil and the 4′-demethylepipodophyllotoxin-4-O-ester of Part B ofthis Example in DMF is stirred for 30 minutes at room temperature. Afterthe mixture is cooled to 0° C., a solution ofdibenzylphosphorochloridate in toluene is added drop by drop. Thesolution is stirred at room temperature for 15 minutes and then dilutedwith cold water and extracted with ether. The ether solution is washedwith water, dried, evaporated under reduced pressure to give the4′-dibenzylphosphate derivative of the compound of part B. A solution ofthe dibenzylphosphate derivative in 85% ethanol is hydrogenated in aParr apparatus in the presence of 10% Pd supported on carbon. Aftertheoretical absorption of hydrogen, the catalyst is filtered, washedwith cold water and suspended in 2N NH₄OH at 50° C. The suspension isfiltered, washed with water and concentrated at reduced pressure at 50°C. The solution is then filtered and acidified with HCl to give thetitle compound.

Example 3 A. Podophyllotoxin-4-O-ester of 4-iodophenoxyacetic acid(000614)

The mixture of podophyllotoxin (41 mg, 0.1 mmol), 4-iodophenoxyaceticacid (55 mg, 0.2 mmol), EDCI (40 mg, 0.14 mmol), DMAP (2 mg, 0.02 mmol)and dichloromethane (5 ml) were stirred in the room temperature for 20h, then dichloromethane (20 ml) was added to the solution. Organic layerwas washed with water (20 ml), saturated NaHCO₃ aqueous solution (10 ml)and brine (20 ml), and then dried over MgSO₄. After the solvent wasremoved under reduced pressure, the resulting liquid was separated bycolumn chromatography (eluent: ethyl acetate and petroleum ether) toafford 35 mg podophyllotoxin-4-O-4-iodophenoxyacetate, mp.

The chemical structure analysis was performed by ¹HNMR (CDCl₃, 600 MHz);δ 7.60 (t, 2H, Ar—H), 6.69 (d, 2H, Ar—H), 6.61 (s, 1H, Ar—H), 6.53 (s,1H, Ar—H), 6.36 (s, 2H, Ar—H), 5.98 (d, 3H, OCH₂O), 4.74 (q, 2H,COCH₂O), 4.60 (d, 1H, H4), 4.34 (t, 1H, H11), 4.20 (t, 1H, H11), 3.81(s, 3H, OCH₃), 3.74 (s, 6H, OCH₃), 2.91 (d, 1H, H2), 2.85 (m, 1H, H3).

B. 4′-demethylepipodophyllotoxin-4-O-ester of 4-iodophenoxyacetic acid

By following the procedure of Part A of this Example, but substituting4′-demethylepipodophyllotoxin for podophyllotoxin, one obtains thecorresponding 4′-demethylepipodophyllotoxin compound.

C. 4′-Phosphate ester of 4′-demethylepipodophyllotoxin-4-O-ester of4-iodophenoxyacetic acid

The compound prepared in part B of this Example is converted to thecorresponding 4′-phosphate ester as follows: A suspension of 50% NaH inmineral oil and the 4′-demethylepipodophyllotoxin-4-O-ester of Part B ofthis Example in DMF is stirred for 30 minutes at room temperature. Afterthe mixture is cooled to 0° C., a solution ofdibenzylphosphorochloridate in toluene is added drop by drop. Thesolution is stirred at room temperature for 15 minutes and then dilutedwith cold water and extracted with ether. The ether solution is washedwith water, dried, evaporated under reduced pressure to give the4′-dibenzylphosphate derivative of the compound of part B. A solution ofthe dibenzylphosphate derivative in 85% ethanol is hydrogenated in aParr apparatus in the presence of 10% Pd supported on carbon. Aftertheoretical absorption of hydrogen, the catalyst is filtered, washedwith cold water and suspended in 2N NH₄OH at 50° C. The suspension isfiltered, washed with water and concentrated at reduced pressure at 50°C. The solution is then filtered and acidified with HCl to give thetitle compound.

Example 4 A. Podophyllotoxin-4-O-ester of 3-chlorophenoxyacetic acid(000622)

The mixture of podophyllotoxin (20 mg, 0.048 mmol),3-chlorophenoxyacetic acid (18 mg, 0.1 mmol), EDCI (30 mg, 0.16 mmol),DMAP (2 mg, 0.02 mmol) and dichloromethane (5 ml) were stirred in theroom temperature for 20 h, then dichloromethane (20 ml) was added to thesolution. Organic later was washed with water (20 ml), saturated NaHCO₃aqueous solution (10 ml) and brine (20 ml), and then dried over MgSO₄.After the solvent was removed under reduced pressure, the resultingliquid was separated by column chromatography (eluent: ethyl acetate andpetroleum ether) to afford 13 mgpodophyllotoxin-4-O-3-chlorophenoxyacetate, mp.

The chemical structure analysis was performed by ¹HNMR (CDCl₃, 600 MHz);δ 7.24 (t, 1H, Ar—H), 7.02 (d, 1H, Ar—H), 6.91 (s, 1H, Ar—H), 6.80 (d,1H, Ar—H), 6.62 (s, 1H, Ar—H), 6.54 (s, 1H, Ar—H), 6.37 (s, 2H, Ar—H),5.98 (d, 3H, OCH₂O, 4.74 (q, 2H, COCH₂O), 4.60 (d, 1H, H4), 4.34 (t, 1H,H11), 4.20 (t, 1H, H11), 3.81 (s, 3H, OCH₃), 3.74 (s, 6H, OCH₃), 2.92(d, 1H, H2), 2.85 (m, 1H, H3).

B. 4′-demethylepipodophyllotoxin-4-O-ester of 3-chlorophenoxyacetic acid

By following the procedure of Part A of this Example, but substituting4′-demethylepipodophyllotoxin for podophyllotoxin, one obtains thecorresponding 4′-demethylepipodophyllotoxin compound.

C. 4′-Phosphate ester of 4′-demethylepipodophyllotoxin-4-O-ester of3-chlorophenoxyacetic acid

The compound prepared in part B of this Example is converted to thecorresponding 4′-phosphate ester as follows: A suspension of 50% NaH inmineral oil and the 4′-demethylepipodophyllotoxin-4-O-ester of Part B ofthis Example in DMF is stirred for 30 minutes at room temperature. Afterthe mixture is cooled to 0° C., a solution ofdibenzylphosphorochloridate in toluene is added drop by drop. Thesolution is stirred at room temperature for 15 minutes and then dilutedwith cold water and extracted with ether. The ether solution is washedwith water, dried, evaporated under reduced pressure to give the4′-dibenzylphosphate derivative of the compound of part B. A solution ofthe dibenzylphosphate derivative in 85% ethanol is hydrogenated in aParr apparatus in the presence of 10% Pd supported on carbon. Aftertheoretical absorption of hydrogen, the catalyst is filtered, washedwith cold water and suspended in 2N NH₄OH at 50° C. The suspension isfiltered, washed with water and concentrated at reduced pressure at 50°C. The solution is then filtered and acidified with HCl to give thetitle compound.

Example 5 A. Podophyllotoxin-4-O-ester of 4-chloro-2-methylphenoxyaceticacid (000317)

The mixture of podophyllotoxin (20 mg, 0.048 mmol),4-chloro-2-methylphenoxyacetic acid (19 mg, 0.1 mmol), EDCI (25 mg, 0.13mmol), DMAP (2 mg, 0.02 mmol) and dichloromethane (3 ml) were stirred inthe room temperature for 20 h, then dichloromethane (20 ml) was added tothe solution. Organic layer was washed with water (20 ml), saturatedNaHCO₃ aqueous solution (10 ml) and brine (20 ml), and then dried overMgSO₄. After the solvent was removed under reduced pressure, theresulting liquid was separated by column chromatography (eluent: ethylacetate and petroleum ether) to afford 12 mgpodophyllotoxin-4-O-4-chloro-2-methylphenoxyacetate, mp.

The chemical structure analysis was performed by ¹HNMR (CDCl₃, 600 MHz);δ 7.13 (t, 2H, Ar—H), 6.66 (d, 1H, Ar—H), 6.59 (s, 1H, Ar—H), 6.54 (s,1H, Ar—H), 6.37 (s, 2H, Ar—H), 5.99 (d, 3H, OCH₂O), 4.76 (q, 2H,COCH₂O), 4.60 (d, 1H, H4), 4.34 (t, 1H, H11), 4.20 (t, 1H, H11), 3.81(s, 3H, OCH₃), 3.74 (s, 6H, OCH₃), 2.95 (d, 1H, H2), 2.85 (m, 1H, H3),2.32 (s, 3H, Ar—CH₃).

B. 4′-demethylepipodophyllotoxin-4-O-ester of4-chloro-2-methylphenoxyacetic acid

By following the procedure of Part A of this Example, but substituting4′-demethylepipodophyllotoxin for podophyllotoxin, one obtains thecorresponding 4′-demethylepipodophyllotoxin compound.

C. 4′-Phosphate ester of 4′-demethylepipodophyllotoxin-4-O-ester of4-chloro-2-methylphenoxyacetic acid

The compound prepared in part B of this Example is converted to thecorresponding 4′-phosphate ester as follows: A suspension of 50% NaH inmineral oil and the 4′-demethylepipodophyllotoxin-4-O-ester of Part B ofthis Example in DMF is stirred for 30 minutes at room temperature. Afterthe mixture is cooled to 0° C., a solution ofdibenzylphosphorochloridate in toluene is added drop by drop. Thesolution is stirred at room temperature for 15 minutes and then dilutedwith cold water and extracted with ether. The ether solution is washedwith water, dried, evaporated under reduced pressure to give the4′-dibenzylphosphate derivative of the compound of part B. A solution ofthe dibenzylphosphate derivative in 85% ethanol is hydrogenated in aParr apparatus in the presence of 10% Pd supported on carbon. Aftertheoretical absorption of hydrogen, the catalyst is filtered, washedwith cold water and suspended in 2N NH₄OH at 50° C. The suspension isfiltered, washed with water and concentrated at reduced pressure at 50°C. The solution is then filtered and acidified with HCl to give thetitle compound.

Example 6 A. Podophyllotoxin-4-O-ester of 4-formylphenoxyacetic acid(000323)

The mixture of podophyllotoxin (20 mg, 0.048 mmol),4-formylphenoxyacetic acid (11 mg, 0.061 mmol), EDCI (25 mg, 0.13 mmol),DMAP (2 mg, 0.02 mmol) and dichloromethane (3 ml) were stirred in theroom temperature for 20 h, then dichlormethane (20 ml) was added to thesolution. Organic layer was washed with water (20 ml), saturated NaHCO₃aqueous solution (10 ml) and brine (20 ml), and then dried over MgSO₄.After the solvent was removed under reduced pressure, the resultingliquid was separated by column chromatography (eluent: ethyl acetate andpetroleum ether) to afford 15 mgpodophyllotoxin-4-O-4-formlyphenoxyacetate, mp.

The chemical structure analysis was performed by ¹HNMR (CDCl₃, 600 MHz);δ 9.92 (s, 1H, CHO), 7.82 (d, 2H, Ar—H), 7.03 (d, 1H, Ar—H), 6.50 (d,2H, Ar—H), 6.36 (s, 2H, Ar-11), 5.99 (d, 3H, OCH₂O), 4.86 (q, 2H,COCH₂O), 4.60 (d, 1H, H4), 4.40 (t, 1H, H11), 4.22 (t, 1H, H11), 3.81(s, 3H, OCH₃), 3.74 (s, 6H, OCH₃), 2.95 (d, 1H, H2), 2.85 (m, 1H, H3).

B. 4′-demethylepipodophyllotoxin-4-O-ester of 4-formylphenoxyacetic acid

By following the procedure of Part A of this Example, but substituting4′-demethylepipodophyllotoxin for podophyllotoxin, one obtains thecorresponding 4′-demethylepipodophyllotoxin compound.

C. 4′-Phosphate ester of 4′-demethylepipodophyllotoxin-4-O-ester of4-formylphenoxyacetic acid

The compound prepared in part B of this Example is converted to thecorresponding 4′-phosphate ester as follows: A suspension of 50% NaH inmineral oil and the 4′-demethylepipodophyllotoxin-4-O-ester of Part B ofthis Example in DMF is stirred for 30 minutes at room temperature. Afterthe mixture is cooled to 0° C., a solution ofdibenzylphosphorochloridate in toluene is added drop by drop. Thesolution is stirred at room temperature for 15 minutes and then dilutedwith cold water and extracted with ether. The ether solution is washedwith water, dried, evaporated under reduced pressure to give the4′-dibenzylphosphate derivative of the compound of part B. A solution ofthe dibenzylphosphate derivative in 85% ethanol is hydrogenated in aParr apparatus in the presence of 10% Pd supported on carbon. Aftertheoretical absorption of hydrogen, the catalyst is filtered, washedwith cold water and suspended in 2N NH₄OH at 50° C. The suspension isfiltered, washed with water and concentrated at reduced pressure at 50°C. The solution is then filtered and acidified with HCl to give thetitle compound.

Example 7 A. Podophyllotoxin-4-O-ester of 4-methoxyphenoxyacetic acid(000329)

The mixture of podophyllotoxin (20 mg, 0.048 mmol),4-formylphenoxyacetic acid (17.4 mg, 0.096 mmol), EDCI (25 mg, 0.13mmol), DMAP (2 mg, 0.02 mmol) and dichloromethane (3 ml) were stirred inthe room temperature for 20 h, then dichloromethane (20 ml) was added tothe solution. Organic layer was washed with water (20 ml), saturatedNaHCO₃ aqueous solution (10 ml) and brine (20 ml), and then dried overMgSO₄. After the solvent was removed under reduced pressure, theresulting liquid was separated by column chromatography (eluent: ethylacetate and petroleum ether) to afford 12 mgpodophyllotoxin-4-O-4-formylphenoxyacetate, mp.

The chemical structure analysis was performed by ¹HNMR (CDCl₃, 600 MHz):δ 6.85 (d, 4H, Ar—H), 6.64 (s, 1H, Ar—H), 6.54 (s, 2H, Ar—H), 6.37 (s,2H, Ar—H), 5.99 (d, 3H, OCH₂O), 4.70 (q, 2H, COCH₂O), 4.60 (d, 1H, H4),4.34 (t, 1H, H11), 4.20 (t, 1H, H11), 3.81 (s, 3H, OCH₃), 3.77 (s, 3H,OCH₃), 3.74 (s, 6H, OCH₃), 2.92 (d, 1H, H2), 2.85 (m, 1H, H3).

B. 4′-demethylepipodophyllotoxin-4-O-ester of 4-methoxyphenoxyaceticacid

By following the procedure of Part A of this Example, but substituting4′-demethylepipodophyllotoxin for podophyllotoxin, one obtains thecorresponding 4′-demethylepipodophyllotoxin compound.

C. 4′-Phosphate ester of 4′-demethylepipodophyllotoxin-4-O-ester of4-methoxyphenoxyacetic acid

The compound prepared in part B of this Example is converted to thecorresponding 4′-phosphate ester as follows: A suspension of 50% NaH inmineral oil and the 4′-demethylepipodophyllotoxin-4-O-ester of Part B ofthis Example in DMF is stirred for 30 minutes at room temperature. Afterthe mixture is cooled to 0° C., a solution ofdibenzylphosphorochloridate in toluene is added drop by drop. Thesolution is stirred at room temperature for 15 minutes and then dilutedwith cold water and extracted with ether. The ether solution is washedwith water, dried, evaporated under reduced pressure to give the4′-dibenzylphosphate derivative of the compound of part B. A solution ofthe dibenzylphosphate derivative in 85% ethanol is hydrogenated in aParr apparatus in the presence of 10% Pd supported on carbon. Aftertheoretical absorption of hydrogen, the catalyst is filtered, washedwith cold water and suspended in 2N NH₄OH at 50° C. The suspension isfiltered, washed with water and concentrated at reduced pressure at 50°C. The solution is then filtered and acidified with HCl to give thetitle compound.

Example 8 A. Podophyllotoxin-4-O-ester of 2,4-dichlorophenoxyacetic acid(000324)

The mixture of podophyllotoxin (20 mg, 0.048 mmol),4-formylphenoxyacetic acid (21.2 mg, 0.096 mmol), EDCI (25 mg, 0.13mmol), DMAP (2 mg, 0.02 mmol) and dichloromethane (3 ml) were stirred inthe room temperature for 20 h, then dichloromethane (20 ml) was added tothe solution. Organic layer was washed with water (20 ml), saturatedNaHCO₃ aqueous solution (10 ml) and brine (20 ml), and then dried overMgSO₄. After the solvent was removed under reduced pressure, theresulting liquid was separated by column chromatography (eluent: ethylacetate and petroleum ether) to afford 13 mgpodophyllotoxin-4-O-2,4-dichlorophenoxyacetate, mp.

The chemical structure analysis was performed by ¹HNMR (CDCl₃, 600 MHz):δ 7.41 (d, 1H, Ar—H), 7.22 (t, 1H, Ar—H), 6.85 (d, 1H, Ar—H), 6.59 (s,1H, Ar—H), 6.54 (s, 1H, Ar—H), 6.37 (s, 2H, Ar—H), 5.99 (d, 2H, OCH₂O),4.81 (q, 2H, COCH₂O), 4.61 (d, 1H, H4), 4.36 (t, 1H, H11), 4.20 (t, 1H,H11), 4.15 (d, 1H, H1), 3.81 (s, 3H, OCH₃), 3.74 (s, 6H, OCH₃), 2.92 (d,1H, H2), 2.85 (m, 1H, H3).

B. 4′-demethylepipodophyllotoxin-4-O-ester of 2,4-dichlorophenoxyaceticacid

By following the procedure of Part A of this Example, but substituting4′-demethylepipodophyllotoxin for podophyllotoxin, one obtains thecorresponding 4′-demethylepipodophyllotoxin compound.

C. 4′-Phosphate ester of 4′-demethylepipodophyllotoxin-4-O-ester of2,4-dichlorophenoxyacetic acid

The compound prepared in part B of this Example is converted to thecorresponding 4′-phosphate ester as follows: A suspension of 50% NaH inmineral oil and the 4′-demethylepipodophyllotoxin-4-O-ester of Part B ofthis Example in DMF is stirred for 30 minutes at room temperature. Afterthe mixture is cooled to 0° C., a solution ofdibenzylphosphorochloridate in toluene is added drop by drop. Thesolution is stirred at room temperature for 15 minutes and then dilutedwith cold water and extracted with ether. The ether solution is washedwith water, dried, evaporated under reduced pressure to give the4′-dibenzylphosphate derivative of the compound of part B. A solution ofthe dibenzylphosphate derivative in 85% ethanol is hydrogenated in aParr apparatus in the presence of 10% Pd supported on carbon. Aftertheoretical absorption of hydrogen, the catalyst is filtered, washedwith cold water and suspended in 2N NH₄OH at 50° C. The suspension isfiltered, washed with water and concentrated at reduced pressure at 50°C. The solution is then filtered and acidified with HCl to give thetitle compound.

Example 9 A. Podophyllotoxin-4-O-ester of7-(carboxymethoxy)-3-chloro-4-methylcoumarin (000405)

The mixture of podophyllotoxin (20 mg, 0.048 mmol),7-(carboxymethoxy)-3-chloro-4-methylcoumarin (25.8 mg, 0.096 mmol), EDCI(25 mg, 0.13 mmol), DMAP (2 mg, 0.02 mmol) and dichloromethane (3 ml)were stirred in the room temperature for 20 h, then dichloromethane (20ml) was added to the solution. Organic layer was washed with water (20ml), saturated NaHCO₃ aqueous solution (10 ml) and brine (20 ml), andthen dried over MgSO₄. After the solvent was removed under reducedpressure, the resulting liquid was separated by column chromatography(eluent: ethyl acetate and petroleum ether) to afford 16 mgpodophyllotoxin-4-O-7-(3-chloro-4-methlycoumarin-7-oxyacetate), mp.

The chemical structure analysis was performed by ¹HNMR (CDCl₃, 600 MHz):δ 7.59 (t, 1H, Ar—H), 6.95 (d, 1H, Ar—H), 6.84 (d, 1H, Ar—H), 6.53 (d,2H, Ar—H), 6.36 (d, 2H, Ar—H), 5.99 (m, 2H, OCH₂O), 4.85 (s, 2H,COCH₂O), 4.61 (s, 1H, H4), 4.38 (t, 1H, H11), 4.20 (t, 1H, H11), 4.15(d, 1H, H1), 3.81 (s, 3H, OCH₃), 3.74 (s, 6H, OCH₃) 2.92 (d, 1H, H2),2.85 (m, 1H, H3), 2.56 (s, 3H, Ar—CH₃).

B. 4′-demethylepipodophyllotoxin-4-O-ester of7-(carboxymethoxy)-3-chloro-4-methylcoumarin

By following the procedure of Part A of this Example, but substituting4′-demethylepipodophyllotoxin for podophyllotoxin, one obtains thecorresponding 4′-demethylepipodophyllotoxin compound.

C. 4′-Phosphate ester of 4′-demethylepipodophyllotoxin-4-O-ester of7-(carboxymethoxy)-3-chloro-4-methylcoumarin

The compound prepared in part B of this Example is converted to thecorresponding 4′-phosphate ester as follows: A suspension of 50% NaH inmineral oil and the 4′-demethylepipodophyllotoxin-4-O-ester of Part B ofthis Example in DMF is stirred for 30 minutes at room temperature. Afterthe mixture is cooled to 0° C., a solution ofdibenzylphosphorochloridate in toluene is added drop by drop. Thesolution is stirred at room temperature for 15 minutes and then dilutedwith cold water and extracted with ether. The ether solution is washedwith water, dried, evaporated under reduced pressure to give the4′-dibenzylphosphate derivative of the compound of part B. A solution ofthe dibenzylphosphate derivative in 85% ethanol is hydrogenated in aParr apparatus in the presence of 10% Pd supported on carbon. Aftertheoretical absorption of hydrogen, the catalyst is filtered, washedwith cold water and suspended in 2N NH₄OH at 50° C. The suspension isfiltered, washed with water and concentrated at reduced pressure at 50°C. The solution is then filtered and acidified with HCl to give thetitle compound.

Example 10 A. Podophyllotoxin-4-O-ester of 4-(4-dichloroethylamino)phenylbutyric acid (003132)

The mixture of podophyllotoxin (10 mg, 0.024 mmol), chlorambucil (11 mg,0.036 mmol), EDCI (25 mg, 0.13 mmol), DMAP (2 mg, 0.02 mmol) anddichloromethane (3 ml) were stirred at 5° C. for 15 h, thendichloromethane (20 ml) was added to the solution. Organic layer waswashed with water (20 ml), saturated NaHCO₃ aqueous solution (10 ml) andbrine (20 ml), and then dried over MgSO₄. After the solvent was removedunder reduced pressure, the resulting liquid was separated by columnchromatography (eluent: ethyl acetate and petroleum ether) to afford 5mg podophyllotoxin-4-O—[4-(4-dichloroethylamino)phenylbutyrate], mp.

The chemical structure analysis was performed by ¹HNMR (CDCl₃, 600 MHz);δ 7.08 (d, 2H, Ar—H), 6.75 (s, 1H, Ar—H), 6.63 (d, 2H, Ar—H), 6.53 (s,1H, Ar—H), 6.39 (s, 2H, Ar—H), 5.99 (d, 2H, OCH₂O), 5.85 (d, 1H,), 4.61(s, H, H4), 4.35 (t, 1H, H11), 4.20 (t, 1H, H11), 3.81 (s, 3H, OCH₃),3.75 (s, 6H, OCH₃), 2.92 (d, 1H, H2), 2.82 (m, 1H, H3), 2.56 (t, 2H,Ar—CH₂), 2.45 (m, 2H, COCH₂), 1.95 (m, 2H, CH₂).

B. 4′-demethylepipodophyllotoxin-4-O-ester of 4-(4-dichloroethylamino)phenylbutyric acid

By following the procedure of Part A of this Example, but substituting4′-demethylepipodophyllotoxin for podophyllotoxin, one obtains thecorresponding 4′-demethylepipodophyllotoxin compound.

C. 4′-Phosphate ester of 4′-demethylepipodophyllotoxin-4-O-ester of4-(4-dichloroethylamino) phenylbutyric acid

The compound prepared in part B of this Example is converted to thecorresponding 4′-phosphate ester as follows: A suspension of 50% NaH inmineral oil and the 4′-demethylepipodophyllotoxin-4-O-ester of Part B ofthis Example in DMF is stirred for 30 minutes at room temperature. Afterthe mixture is cooled to 0° C., a solution ofdibenzylphosphorochloridate in toluene is added drop by drop. Thesolution is stirred at room temperature for 15 minutes and then dilutedwith cold water and extracted with ether. The ether solution is washedwith water, dried, evaporated under reduced pressure to give the4′-dibenzylphosphate derivative of the compound of part B. A solution ofthe dibenzylphosphate derivative in 85% ethanol is hydrogenated in aParr apparatus in the presence of 10% Pd supported on carbon. Aftertheoretical absorption of hydrogen, the catalyst is filtered, washedwith cold water and suspended in 2N NH₄OH at 50° C. The suspension isfiltered, washed with water and concentrated at reduced pressure at 50°C. The solution is then filtered and acidified with HCl to give thetitle compound.

Example 11 A. Podophyllotoxin-4-O-ester of 3-chloroacetamidobenzoic acid(000124)

The mixture of podophyllotoxin (10 mg, 0.024 mmol),3-chloroacetamidobenzoic acid (12.5 mg, 0.05 mmol), EDCI (25 mg, 0.13mmol), DMAP (2 mg, 0.02 mmol) and dichloromethane (3 ml) were stirred atroom temperature for 22 h, then dichloromethane (20 ml) was added to thesolution. Organic layer was washed with water (20 ml), saturated NaHCO₃aqueous solution (10 ml) and brine (20 ml), and then dried over MgSO₄.After the solvent was removed under reduced pressure, the resultingliquid was separated by column chromatography (eluent: ethyl acetate andpetroleum ether) to afford 6 mgpodophyllotoxin-4-O—[3-chloroacetamidobenzoate], mp.

The chemical structure analysis was performed by ¹HNMR (CDCl₃, 600 MHz):δ 8.36 (s, 1H, Ar—H), 8.17 (s, 1H, Ar—H), 7.90 (d, 2H, Ar—H), 7.55 (s,1H, Ar—H), 6.86 (s, 1H, Ar—H), 6.59 (s, 1H, Ar—H), 6.45 (s, 2H, Ar—H),6.14 (s, 1H), 6.00 (s, 2H, OCH₂O), 4.65 (s, 1H, H4), 4.44 (s, 1H, H11),4.31 (t, 1H, H11), 4.21 (s, 2H, CICH₂CO), 3.79 (s, 9H, OCH₃), 3.00 (s,2H, H2,3).

B. 4′-demethylepipodophyllotoxin-4-O-ester of 3-chloroacetamidobenzoicacid

By following the procedure of Part A of this Example, but substituting4′-demethylepipodophyllotoxin for podophyllotoxin, one obtains thecorresponding 4′-demethylepipodophyllotoxin compound.

C. 4′-Phosphate ester of 4′-demethylepipodophyllotoxin-4-O-ester of3-chloroacetamidobenzoic acid

The compound prepared in part B of this Example is converted to thecorresponding 4′-phosphate ester as follows: A suspension of 50% NaH inmineral oil and the 4′-demethylepipodophyllotoxin-4-O-ester of Part B ofthis Example in DMF is stirred for 30 minutes at room temperature. Afterthe mixture is cooled to 0° C., a solution ofdibenzylphosphorochloridate in toluene is added drop by drop. Thesolution is stirred at room temperature for 15 minutes and then dilutedwith cold water and extracted with ether. The ether solution is washedwith water, dried, evaporated under reduced pressure to give the4′-dibenzylphosphate derivative of the compound of part B. A solution ofthe dibenzylphosphate derivative in 85% ethanol is hydrogenated in aParr apparatus in the presence of 10% Pd supported on carbon. Aftertheoretical absorption of hydrogen, the catalyst is filtered, washedwith cold water and suspended in 2N NH₄OH at 50° C. The suspension isfiltered, washed with water and concentrated at reduced pressure at 50°C. The solution is then filtered and acidified with HCl to give thetitle compound.

Example 12 A. Podophyllotoxin-4-O-ester of chromone-2-carboxylic acid(000215)

The mixture of podophyllotoxin (20 mg, 0.048 mmol),chromone-2-carboxylic acid (19 mg, 0.1 mmol), EDCI (25 mg, 0.13 mmol),DMAP (2 mg, 0.02 mmol) and dichloromethane (3 ml) were stirred at roomtemperature for 15 h, then dichloromethane (20 ml) was added to thesolution. Organic layer was washed with water (20 ml), saturated NaHCO₃aqueous solution (10 ml) and brine (20 ml), and then dried over MgSO₄.After the solvent was removed under reduced pressure, the resultingliquid was separated by column chromatography (eluent: ethyl acetate andpetroleum ether) to afford 10 mgpodophyllotoxin-4-O-chromone-2-carboxylate, mp.

The chemical structure analysis was performed by ¹HNMR (CDCl₃, 600 MHz):δ 8.22 (s, 1H, Ar—H), 7.78 (s, 1H, Ar—H), 7.58 (s, 1H, Ar—H), 7.49 (s,1H, Ar—H), 7.15 (s, 1H, Ar—H), 6.83 (s, 1H, Ar—H), 6.61 (s, 1H, Ar—H),6.42 (s, 2H, Ar—H), 6.15 (s, 1H), 6.03 (s, 2H, OCH₂O), 4.67 (s, 1H,),4.49 (s, 1H, H4), 4.33 (t, 1H, H11), 4.20 (t, 1H, H11), 3.79 (s, 9H,OCH₃), 3.02 (s, 2H, H2, 3).

B. 4′-demethylepipodophyllotoxin-4-O-ester of chromone-2-carboxylic acid

By following the procedure of Part A of this Example, but substituting4′-demethylepipodophyllotoxin for podophyllotoxin, one obtains thecorresponding 4′-demethylepipodophyllotoxin compound.

C. 4′-Phosphate ester of 4′-demethylepipodophyllotoxin-4-O-ester ofchromone-2-carboxylic acid

The compound prepared in part B of this Example is converted to thecorresponding 4′-phosphate ester as follows: A suspension of 50% NaH inmineral oil and the 4′-demethylepipodophyllotoxin-4-O-ester of Part B ofthis Example in DMF is stirred for 30 minutes at room temperature. Afterthe mixture is cooled to 0° C., a solution ofdibenzylphosphorochloridate in toluene is added drop by drop. Thesolution is stirred at room temperature for 15 minutes and then dilutedwith cold water and extracted with ether. The ether solution is washedwith water, dried, evaporated under reduced pressure to give the4′-dibenzylphosphate derivative of the compound of part B. A solution ofthe dibenzylphosphate derivative in 85% ethanol is hydrogenated in aParr apparatus in the presence of 10% Pd supported on carbon. Aftertheoretical absorption of hydrogen, the catalyst is filtered, washedwith cold water and suspended in 2N NH₄OH at 50° C. The suspension isfiltered, washed with water and concentrated at reduced pressure at 50°C. The solution is then filtered and acidified with HCl to give thetitle compound.

Example 13 A. Podophyllotoxin-4-O-ester of 5-nitro-2-furoic acid(000202)

The mixture of podophyllotoxin (20 mg, 0.048 mmol), 5-nitro-2-furoicacid (30 mg, 0.2 mmol), EDCI (38 mg, 0.2 mmol), DMAP (2 mg, 0.02 mmol)and dichloromethane (3 ml) were stirred at room temperature for 20 h,then dichloromethane (20 ml) was added to the solution. Organic layerwas washed with water (20 ml), saturated NaHCO₃ aqueous solution (10 ml)and brine (20 ml), and then dried over MgSO₄. After the solvent wasremoved under reduced pressure, the resulting liquid was separated bycolumn chromatography (eluent: ethyl acetate and petroleum ether) toafford 18.2 mg podophyllotoxin-4-O-[5-nitro-s-furoate], mp.

The chemical structure analysis was performed by ¹HNMR (CDCl₃, 600 MHz);δ 7.38 (s, 2H, Ar—H), 6.81 (s, 1H, Ar—H), 6.61 (s, 1H, Ar—H), 6.48 (s,2H, Ar—H), 6.19 (d, 1H), 6.01 (t, 2H, OCH₂O) 4.65 (s, 1H,), 4.45 (t, 1H,H4), 4.33 (t, 1H, H11), 3.80 (s, 9H, OCH₃), 3.01 (m, 2H, H2,3).

B. 4′-demethylepipodophyllotoxin-4-O-ester of 5-nitro-2-furoic acid

By following the procedure of Part A of this Example, but substituting4′-demethylepipodophyllotoxin for podophyllotoxin, one obtains thecorresponding 4′-demethylepipodophyllotoxin compound.

C. 4′-Phosphate ester of 4′-demethylepipodophyllotoxin-4-O-ester of5-nitro-2-furoic acid

The compound prepared in part B of this Example is converted to thecorresponding 4′-phosphate ester as follows: A suspension of 50% NaH inmineral oil and the 4′-demethylepipodophyllotoxin-4-O-ester of Part B ofthis Example in DMF is stirred for 30 minutes at room temperature. Afterthe mixture is cooled to 0° C., a solution ofdibenzylphosphorochloridate in toluene is added drop by drop. Thesolution is stirred at room temperature for 15 minutes and then dilutedwith cold water and extracted with ether. The ether solution is washedwith water, dried, evaporated under reduced pressure to give the4′-dibenzylphosphate derivative of the compound of part B. A solution ofthe dibenzylphosphate derivative in 85% ethanol is hydrogenated in aParr apparatus in the presence of 10% Pd supported on carbon. Aftertheoretical absorption of hydrogen, the catalyst is filtered, washedwith cold water and suspended in 2N NH₄OH at 50° C. The suspension isfiltered, washed with water and concentrated at reduced pressure at 50°C. The solution is then filtered and acidified with HCl to give thetitle compound.

Example 14 A. Podophyllotoxin-4-O-ester of anthraquinone-2-carboxylicacid (000121)

The mixture of podophyllotoxin (20 mg, 0.048 mmol,anthraquinone-2-carboxylic chloride (27 mg, 0.1 mmol), triethylamine (10mg, 0.1 mmol), and dichloromethane (3 ml) were stirred at roomtemperature for 20 h, then dichlormethane (20 ml) was added to thesolution. Organic layer was washed with water (20 ml), saturated NaHCO₃aqueous solution (10 ml) and brine (20 ml), and then dried over MgSO₄.After the solvent was removed under reduced pressure, the resultingliquid was separated by column chromatography (eluent: ethyl acetate andpetroleum ether) to afford 18.2 mgpodophyllotoxin-4-O-anthraquinone-2-carboxylate, mp.

The chemical structure analysis was performed by ¹HNMR (CDCl₃, 600 MHz):δ 8.93 (s, 1H, Ar—H), 8.46 (s, 2H, Ar—H), 8.35 (d, 2H, Ar—H), 7.87 (s,2H, Ar—H), 6.87 (s, 1H, Ar—H), 6.62 (s, 1H, Ar—H), 6.48 (s, 2H, Ar—H),6.23 (d, 1H), 6.01 (d, 2H, OCH₂O), 4.68 (s, 1H,) 4.45 (t, 1H, H4), 4.33(t, 1H, H11), 3.82 (s, 9H, OCH₃), 3.04 (m, 2H, H2, 3).

B. 4′-demethylepipodophyllotoxin-4-O-ester of anthraquinone-2-carboxylicacid

By following the procedure of Part A of this Example, but substituting4′-demethylepipodophyllotoxin for podophyllotoxin, one obtains thecorresponding 4′-demethylepipodophyllotoxin compound.

C. 4′-Phosphate ester of 4′-demethylepipodophyllotoxin-4-O-ester ofanthraquinone-2-carboxylic acid

The compound prepared in part B of this Example is converted to thecorresponding 4′-phosphate ester as follows: A suspension of 50% NaH inmineral oil and the 4′-demethylepipodophyllotoxin-4-O-ester of Part B ofthis Example in DMF is stirred for 30 minutes at room temperature. Afterthe mixture is cooled to 0° C., a solution ofdibenzylphosphorochloridate in toluene is added drop by drop. Thesolution is stirred at room temperature for 15 minutes and then dilutedwith cold water and extracted with ether. The ether solution is washedwith water, dried, evaporated under reduced pressure to give the4′-dibenzylphosphate derivative of the compound of part B. A solution ofthe dibenzylphosphate derivative in 85% ethanol is hydrogenated in aParr apparatus in the presence of 10% Pd supported on carbon. Aftertheoretical absorption of hydrogen, the catalyst is filtered, washedwith cold water and suspended in 2N NH₄OH at 50° C. The suspension isfiltered, washed with water and concentrated at reduced pressure at 50°C. The solution is then filtered and acidified with HCl to give thetitle compound.

Example 15 A. Podophyllotoxin-4-O-ester of2-phenyl-4-quinolinecarboxylic acid (000125)

The mixture of podophyllotoxin (20 mg, 0.048 mmol),2-phenyl-4-quinolinecarboxylic acid (12.5 mg, 0.2 mmol), EDCI (25 mg,0.13 mmol), DMAP (2 mg, 0.02 mmol) and dichloromethane (3 ml) werestirred at room temperature for 20 h, then dichloromethane (20 ml) wasadded to the solution. Organic layer was washed with water (20 ml),saturated NaHCO₃ aqueous solution (10 ml) and brine (20 ml), and thendried over MgSO₄. After the solvent was removed under reduced pressure,the resulting liquid was separated by column chromatography (eluent:ethyl acetate and petroleum ether) to afford 18 mgpodophyllotoxin-4-O—[2-phenyl-4-quinolinecarboxylate], mp.

The chemical structure analysis was performed by ¹HNMR (CDCl₃, 600 MHz):δ 8.78 (d, 1H, Ar—H), 8.40 (s, 1H, Ar—H), 8.28 (d, 1H, Ar—H), 8.14 (d,2H, Ar—H), 7.81 (t, 1H, Ar—H), 7.64 (t, 1H, Ar—H), 7.56 (m, 3H, Ar—H),6.92 (s, 1H, Ar—H), 6.61 (s, 1H, Ar—H), 6.47 (s, 2H, Ar—H), 6.31 (d,1H), 5.99 (t, 2H, OCH₂O), 4.69 (s, 1H,), 4.60 (t, 1H, H4), 4.40 (t, 1H,H11), 3.81 (s, 3H, OCH₃), 3.74 (s, 6H, OCH₃), 3.07 (m, 2H, H2, 3).

B. 4′-demethylepipodophyllotoxin-4-O-ester of2-phenyl-4-quinolinecarboxylic acid

By following the procedure of Part A of this Example, but substituting4′-demethylepipodophyllotoxin for podophyllotoxin, one obtains thecorresponding 4′-demethylepipodophyllotoxin compound.

C. 4′-Phosphate ester of 4′-demethylepipodophyllotoxin-4-O-ester of2-phenyl-4-quinolinecarboxylic acid

The compound prepared in part B of this Example is converted to thecorresponding 4′-phosphate ester as follows: A suspension of 50% NaH inmineral oil and the 4′-demethylepipodophyllotoxin-4-O-ester of Part B ofthis Example in DMF is stirred for 30 minutes at room temperature. Afterthe mixture is cooled to 0° C., a solution ofdibenzylphosphorochloridate in toluene is added drop by drop. Thesolution is stirred at room temperature for 15 minutes and then dilutedwith cold water and extracted with ether. The ether solution is washedwith water, dried, evaporated under reduced pressure to give the4′-dibenzylphosphate derivative of the compound of part B. A solution ofthe dibenzylphosphate derivative in 85% ethanol is hydrogenated in aParr apparatus in the presence of 10% Pd supported on carbon. Aftertheoretical absorption of hydrogen, the catalyst is filtered, washedwith cold water and suspended in 2N NH₄OH at 50° C. The suspension isfiltered, washed with water and concentrated at reduced pressure at 50°C. The solution is then filtered and acidified with HCl to give thetitle compound.

Example 16 A. Podophyllotoxin-4-O-ester of thymine-1-acetic acid(0003061)

The mixture of podophyllotoxin (20 mg, 0.048 mmol), thymine-1-aceticacid (18 mg, 0.1 mmol), EDCI (25 mg, 0.13 mmol), DMAP (2 mg, 0.02 mmol),DMF (2 ml) and dichloromethane (2 ml) were stirred at room temperaturefor 20 h, then dichloromethane (20 ml) was added to the solution.Organic layer was washed with water (20 ml), saturated NaHCO₃ aqueoussolution (10 ml) and brine (20 ml), and then dried over MgSO₄. After thesolvent was removed under reduced pressure, the resulting liquid wasseparated by column chromatography (eluent: ethyl acetate and petroleumether) to afford 18 mg podophyllotoxin-4-O—[thymine-1-acetate], mp.

The chemical structure analysis was performed by ¹HNMR (CDCl₃, 600 MHz):δ 9.22 (s, 1H, NH), 7.02 (s, 1H, Ar—H), 6.79 (s, 1H, Ar—H), 6.54 (s, 1H,Ar—H), 6.36 (s, 1H, Ar—H), 6.01 (s, 2H, Ar—H), 5.99 (t, 3H, OCH₂O), 4.60(s, 1H, H4), 4.52 (q, 2H, COCH₂N), 4.40 (t, 1H, H11), 4.20 (t, 1H), 3.81(s, 3H, OCH₃), 3.74 (s, 6H, OCH₃), 3.07 (m, 2H, H2,3), 1.93 (s, 3H,Ar—H).

B. 4′-demethylepipodophyllotoxin-4-O-ester of thymine-1-acetic acid

By following the procedure of Part A of this Example, but substituting4′-demethylepipodophyllotoxin for podophyllotoxin, one obtains thecorresponding 4′-demethylepipodophyllotoxin compound.

C. 4′-Phosphate ester of 4′-demethylepipodophyllotoxin-4-O-ester ofthymine-1-acetic acid

The compound prepared in part B of this Example is converted to thecorresponding 4′-phosphate ester as follows: A suspension of 50% NaH inmineral oil and the 4′-demethylepipodophyllotoxin-4-O-ester of Part B ofthis Example in DMF is stirred for 30 minutes at room temperature. Afterthe mixture is cooled to 0° C., a solution ofdibenzylphosphorochloridate in toluene is added drop by drop. Thesolution is stirred at room temperature for 15 minutes and then dilutedwith cold water and extracted with ether. The ether solution is washedwith water, dried, evaporated under reduced pressure to give the4′-dibenzylphosphate derivative of the compound of part B. A solution ofthe dibenzylphosphate derivative in 85% ethanol is hydrogenated in aParr apparatus in the presence of 10% Pd supported on carbon. Aftertheoretical absorption of hydrogen, the catalyst is filtered, washedwith cold water and suspended in 2N NH₄OH at 50° C. The suspension isfiltered, washed with water and concentrated at reduced pressure at 50°C. The solution is then filtered and acidified with HCl to give thetitle compound.

Example 17 A. Podophyllotoxin-4-O-ester of hemisuccinic acid (000201)

The mixture of podophyllotoxin (50 mg, 0.12 mmol), succinic anhydride(33 mg, 0.3 mmol), DMAP (2 mg, 0.02 mmol) and THF (3 ml) were stirred at70° C. for 20 h, then THF was removed and dichloromethane (20 ml) wasadded to the residue. Organic layer was washed with water (20 ml) andbrine (20 ml), and then dried over MgSO₄. After the solvent was removedunder reduced pressure, the resulting liquid was separated by columnchromatography (eluent: ethyl acetate and petroleum ether) to afford 20mg podophyllotoxin-4-O-hemisuccinate, mp.

The chemical structure analysis was performed by ¹HNMR (CDCl₃, 600 MHz):δ 6.78 (s, 1H, Ar—H), 6.53 (s, 1H, Ar—H), 6.39 (s, 2H, Ar—H), 5.98 (t,2H, OCH₂O), 5.92 (d, 1H), 4.60 (s, 1H, H4), 4.40 (t, 1H, H11), 4.17 (t,1H), 3.81 (s, 3H, OCH₃), 3.76 (s, 6H, OCH₃), 2.95 (m, 2H, H2, 3), 2.73(m, 4H, COCH₂CH₂CO).

B. 4′-demethylepipodophyllotoxin-4-O-ester of hemisuccinic acid

By following the procedure of Part A of this Example, but substituting4′-demethylepipodophyllotoxin for podophyllotoxin, one obtains thecorresponding 4′-demethylepipodophyllotoxin compound.

C. 4′-Phosphate ester of 4′-demethylepipodophyllotoxin-4-O-ester ofhemisuccinic acid

The compound prepared in part B of this Example is converted to thecorresponding 4′-phosphate ester as follows: A suspension of 50% NaH inmineral oil and the 4′-demethylepipodophyllotoxin-4-O-ester of Part B ofthis Example in DMF is stirred for 30 minutes at room temperature. Afterthe mixture is cooled to 0° C., a solution ofdibenzylphosphorochloridate in toluene is added drop by drop. Thesolution is stirred at room temperature for 15 minutes and then dilutedwith cold water and extracted with ether. The ether solution is washedwith water, dried, evaporated under reduced pressure to give the4′-dibenzylphosphate derivative of the compound of part B. A solution ofthe dibenzylphosphate derivative in 85% ethanol is hydrogenated in aParr apparatus in the presence of 10% Pd supported on carbon. Aftertheoretical absorption of hydrogen, the catalyst is filtered, washedwith cold water and suspended in 2N NH₄OH at 50° C. The suspension isfiltered, washed with water and concentrated at reduced pressure at 50°C. The solution is then filtered and acidified with HCl to give thetitle compound.

Example 18 A. Bis(Podophyllotoxin-4-O-ester) of 5-nitroisophthalic acid(000331)

The mixture of podophyllotoxin (30 mg, 0.072 mmol), 5-nitroisophthalicacid (7.6 mg, 0.036 mmol), EDCI (25 mg, 0.13 mmol), DMAP (2 mg, 0.02mmol) and dichloromethane (3 ml) were stirred at room temperature for 20h, then dichloromethane (20 ml) was added to the solution. Organic layerwas washed with water (20 ml), saturated NaHCO₃ aqueous solution (10 ml)and brine (20 ml), and then dried over MgSO₄. After the solvent wasremoved under reduced pressure, the resulting liquid was separated bycolumn chromatography (eluent: ethyl acetate and petroleum ether) toafford 15 mg Bis(Podophyllotoxin-4-O-ester) of 5-nitroisophthalic acidmp.

The chemical structure analysis was performed by ¹HNMR (CDCl₃, 600 MHz):δ 9.03 (s, 2H, Ar—H), 6.79 (s, 2H, Ar—H), 6.63 (s, 2H, Ar—H), 6.46 (s,4H, Ar—H), 6.25 (d, 1H), 6.02 (s, 2H, OCH₂O), 4.69 (s, 1H,), 4.43 (t,1H, H11), 4.32 (s, 1H,), 3.81 (s, 9H, OCH₃), 3.04 (m, 2H, H2,3).

B. 4′-demethylepipodophyllotoxin-4-O-ester of 5-nitroisophthalic acid

By following the procedure of Part A of this Example, but substituting4′-demethylepipodophyllotoxin for podophyllotoxin, one obtains thecorresponding 4′-demethylepipodophyllotoxin compound.

C. 4′-Phosphate ester of 4′-demethylepipodophyllotoxin-4-O-ester of5-nitroisophthalic acid

The compound prepared in part B of this Example is converted to thecorresponding 4′-phosphate ester as follows: A suspension of 50% NaH inmineral oil and the 4′-demethylepipodophyllotoxin-4-O-ester of Part B ofthis Example in DMF is stirred for 30 minutes at room temperature. Afterthe mixture is cooled to 0° C., a solution ofdibenzylphosphorochloridate in toluene is added drop by drop. Thesolution is stirred at room temperature for 15 minutes and then dilutedwith cold water and extracted with ether. The ether solution is washedwith water, dried, evaporated under reduced pressure to give the4′-dibenzylphosphate derivative of the compound of part B. A solution ofthe dibenzylphosphate derivative in 85% ethanol is hydrogenated in aParr apparatus in the presence of 10% Pd supported on carbon. Aftertheoretical absorption of hydrogen, the catalyst is filtered, washedwith cold water and suspended in 2N NH₄OH at 50° C. The suspension isfiltered, washed with water and concentrated at reduced pressure at 50°C. The solution is then filtered and acidified with HCl to give thetitle compound.

Example 19 A. Podophyllotoxin-4-O-ester ofN-(tert-Butoxycarbonyl)-L-proline [000203]

The mixture of podophyllotoxin (20 mg, 0.05 mmol),N-(tert-Butoxycarbonyl)-L-proline carboxylic acid (21.5 mg, 0.1 mmol),EDCI (25 mg, 0.13 mmol), DMAP (2 mg, 0.02 mmol) and dichloromethane (3ml) were stirred in the room temperature for 20 h, then dichloromethane(20 ml) was added to the solution. Organic layer was washed with water(20 ml), saturated NaHCO₃ aqueous solution (10 ml) and brine (20 ml),and then dried over MgSO₄. After the solvent was removed under reducedpressure, the resulting liquid was separated by column chromatography(eluent: ethyl acetate and petroleum ether) to afford 3.5 mgPodophyllotoxin-4-O-ester of N-(tert-Butoxycarbonyl)-L-prolinecarboxylic acid.

The chemical structure analysis was performed by ¹HNMR (CDCl₃, 600 MHz):

B. 4′-demethylepipodophyllotoxin-4-O-ester ofN-(tert-Butoxycarbonyl)-L-proline

By following the procedure of Part A of this Example, but substituting4′-demethylepipodophyllotoxin for podophyllotoxin, one obtains thecorresponding 4′-demethylepipodophyllotoxin compound.

C. 4′-Phosphate ester of 4′-demethylepipodophyllotoxin-4-O-ester ofN-(tert-Butoxycarbonyl)-L-proline

The compound prepared in part B of this Example is converted to thecorresponding 4′-phosphate ester as follows: A suspension of 50% NaH inmineral oil and the 4′-demethylepipodophyllotoxin-4-O-ester of Part B ofthis Example in DMF is stirred for 30 minutes at room temperature. Afterthe mixture is cooled to 0° C., a solution ofdibenzylphosphorochloridate in toluene is added drop by drop. Thesolution is stirred at room temperature for 15 minutes and then dilutedwith cold water and extracted with ether. The ether solution is washedwith water, dried, evaporated under reduced pressure to give the4′-dibenzylphosphate derivative of the compound of part B. A solution ofthe dibenzylphosphate derivative in 85% ethanol is hydrogenated in aParr apparatus in the presence of 10% Pd supported on carbon. Aftertheoretical absorption of hydrogen, the catalyst is filtered, washedwith cold water and suspended in 2N NH₄OH at 50° C. The suspension isfiltered, washed with water and concentrated at reduced pressure at 50°C. The solution is then filtered and acidified with HCl to give thetitle compound.

Example 20 A. Podophyllotoxin-4-O-ester of (+)-menthoxyethanoic acid[000218]

The mixture of podophyllotoxin (20 mg, 0.048 mmol), (+)-menthoxyethanoicacid (20.5 mg, 0.1 mmol), EDCI (25 mg, 0.13 mmol), DMAP (2 mg, 0.02mmol) and dichloromethane (3 ml) were stirred in the room temperaturefor 20 h, then dichloromethane (20 ml) was added to the solution.Organic layer was washed with water (20 ml), saturated NaHCO₃ aqueoussolution (10 ml) and brine (20 ml), and then dried over MgSO₄. After thesolvent was removed under reduced pressure, the resulting liquid wasseparated by column chromatography (eluent: ethyl acetate and petroleumether) to afford 12 mg Podophyllotoxin-4-O-ester of (+)-menthoxyethanoicacid.

The chemical structure analysis was performed by ¹HNMR (CDCl₃, 600 MHz):

B. 4′-demethylepipodophyllotoxin-4-O-ester of (+)-menthoxyethanoic acid

By following the procedure of Part A of this Example, but substituting4′-demethylepipodophyllotoxin for podophyllotoxin, one obtains thecorresponding 4′-demethylepipodophyllotoxin compound.

C. 4′-Phosphate ester of 4′-demethylepipodophyllotoxin-4-O-ester of(+)-menthoxyethanoic acid

The compound prepared in part B of this Example is converted to thecorresponding 4′-phosphate ester as follows: A suspension of 50% NaH inmineral oil and the 4′-demethylepipodophyllotoxin-4-O-ester of Part B ofthis Example in DMF is stirred for 30 minutes at room temperature. Afterthe mixture is cooled to 0° C., a solution ofdibenzylphosphorochloridate in toluene is added drop by drop. Thesolution is stirred at room temperature for 15 minutes and then dilutedwith cold water and extracted with ether. The ether solution is washedwith water, dried, evaporated under reduced pressure to give the4′-dibenzylphosphate derivative of the compound of part B. A solution ofthe dibenzylphosphate derivative in 85% ethanol is hydrogenated in aParr apparatus in the presence of 10% Pd supported on carbon. Aftertheoretical absorption of hydrogen, the catalyst is filtered, washedwith cold water and suspended in 2N NH₄OH at 50° C. The suspension isfiltered, washed with water and concentrated at reduced pressure at 50°C. The solution is then filtered and acidified with HCl to give thetitle compound.

Example 21 A. Podophyllotoxin-4-O-ester of(+)-2-(2,4,5,7-tetranitro-9-enylideaminooxy)-propionic acid [000222]

The mixture of podophyllotoxin (20 mg, 0.048 mmol),(+)-2-(2,4,5,7-tetranitro-9-enylideaminooxy)-propionic acid (22 mg,0.048 mmol), EDCI (25 mg, 0.13 mmol), DMAP (2 mg, 0.02 mmol) anddichloromethane (3 ml) were stirred in the room temperature for 20 h,then dichloromethane (20 ml) was added to the solution. Organic layerwas washed with water (20 ml), saturated NaHCO₃ aqueous solution (10 ml)and brine (20 ml), and then dried over MgSO₄. After the solvent wasremoved under reduced pressure, the resulting liquid was separated bycolumn chromatography (eluent: ethyl acetate and petroleum ether) toafford Podophyllotoxin-4-O-ester of(+)-2-(2,4,5,7-tetranitro-9-enylideaminooxy)-propionic acid.

The chemical structure analysis was performed by ¹HNMR (CDCl₃, 600 MHz):

B. 4′-demethylepipodophyllotoxin-4-O-ester of(+)-2-(2,4,5,7-tetranitro-9-enylideaminooxy)-propionic acid

By following the procedure of Part A of this Example, but substituting4′-demethylepipodophyllotoxin for podophyllotoxin, one obtains thecorresponding 4′-demethylepipodophyllotoxin compound.

C. 4′-Phosphate ester of 4′-demethylepipodophyllotoxin-4-O-ester of(+)-2-(2,4,5,7-tetranitro-9-enylideaminooxy)-propionic acid

The compound prepared in part B of this Example is converted to thecorresponding 4′-phosphate ester as follows: A suspension of 50% NaH inmineral oil and the 4′-demethylepipodophyllotoxin-4-O-ester of Part B ofthis Example in DMF is stirred for 30 minutes at room temperature. Afterthe mixture is cooled to 0° C., a solution ofdibenzylphosphorochloridate in toluene is added drop by drop. Thesolution is stirred at room temperature for 15 minutes and then dilutedwith cold water and extracted with ether. The ether solution is washedwith water, dried, evaporated under reduced pressure to give the4′-dibenzylphosphate derivative of the compound of part B. A solution ofthe dibenzylphosphate derivative in 85% ethanol is hydrogenated in aParr apparatus in the presence of 10% Pd supported on carbon. Aftertheoretical absorption of hydrogen, the catalyst is filtered, washedwith cold water and suspended in 2N NH₄OH at 50° C. The suspension isfiltered, washed with water and concentrated at reduced pressure at 50°C. The solution is then filtered and acidified with HCl to give thetitle compound.

Example 22 A. Podophyllotoxin-4-O-ester ofN—BOC-1,2,3,4-Tetrahydro-β-carboline-3-carboxylic acid [000301]

The mixture of podophyllotoxin (20 mg, 0.048 mmol),N—BOC-1,2,3,4-Tetrahydro-β-carboline-3-carboxylic acid (22 mg, 0.07mmol), EDCI (25 mg, 0.13 mmol), DMAP (2 mg, 0.02 mmol) anddichloromethane (3 ml) were stirred in the room temperature for 20 h,then dichloromethane (20 ml) was added to the solution. Organic layerwas washed with water (20 ml), saturated NaHCO₃ aqueous solution (10 ml)and brine (20 ml), and then dried over MgSO₄. After the solvent wasremoved under reduced pressure, the resulting liquid was separated bycolumn chromatography (eluent: ethyl acetate and petroleum ether) toafford 18 mg Podophyllotoxin-4-O-ester ofN—BOC-1,2,3,4-Tetrahydro-β-carboline-3-carboxylic acid

The chemical structure analysis was performed by ¹HNMR (CDCl₃, 600 MHz):

B. 4′-demethylepipodophyllotoxin-4-O-ester ofN—BOC-1,2,3,4-Tetrahydro-β-carboline-3-carboxylic acid

By following the procedure of Part A of this Example, but substituting4′-demethylepipodophyllotoxin for podophyllotoxin, one obtains thecorresponding 4′-demethylepipodophyllotoxin compound.

C. 4′-Phosphate ester of 4′-demethylepipodophyllotoxin-4-O-ester ofN—BOC-1,2,3,4-Tetrahydro-β-carboline-3-carboxylic acid

The compound prepared in part B of this Example is converted to thecorresponding 4′-phosphate ester as follows: A suspension of 50% NaH inmineral oil and the 4′-demethylepipodophyllotoxin-4-O-ester of Part B ofthis Example in DMF is stirred for 30 minutes at room temperature. Afterthe mixture is cooled to 0° C., a solution ofdibenzylphosphorochloridate in toluene is added drop by drop. Thesolution is stirred at room temperature for 15 minutes and then dilutedwith cold water and extracted with ether. The ether solution is washedwith water, dried, evaporated under reduced pressure to give the4′-dibenzylphosphate derivative of the compound of part B. A solution ofthe dibenzylphosphate derivative in 85% ethanol is hydrogenated in aParr apparatus in the presence of 10% Pd supported on carbon. Aftertheoretical absorption of hydrogen, the catalyst is filtered, washedwith cold water and suspended in 2N NH₄OH at 50° C. The suspension isfiltered, washed with water and concentrated at reduced pressure at 50°C. The solution is then filtered and acidified with HCl to give thetitle compound.

Example 23 A. Podophyllotoxin-4-O-ester ofN—BOC-1,2,3,4-Tetrahydroisoquinoline-3-carboxylic acid [000302]

The mixture of podophyllotoxin (20 mg, 0.048 mmol),N—BOC-1,2,3,4-Tetrahydroisoquinoline-3-carboxylic acid (22 mg, 0.07mmol), EDCI (25 mg, 0.13 mmol), DMAP (2 mg, 0.02 mmol) anddichloromethane (3 ml) were stirred in the room temperature for 20 h,then dichloromethane (20 ml) was added to the solution. Organic layerwas washed with water (20 ml), saturated NaHCO₃ aqueous solution (10 ml)and brine (20 ml), and then dried over MgSO₄. After the solvent wasremoved under reduced pressure, the resulting liquid was separated bycolumn chromatography (eluent: ethyl acetate and petroleum ether) toafford 15 mgPodophyllotoxin-4-O-ester ofN—BOC-1,2,3,4-Tetrahydroisoquinoline-3-carboxylic acid.

The chemical structure analysis was performed by ¹HNMR (CDCl₃, 600 MHz):

B. 4′-demethylepipodophyllotoxin-4-O-ester of 4N—BOC-1,2,3,4-Tetrahydroisoquinoline-3-carboxylic acid

By following the procedure of Part A of this Example, but substituting4′-demethylepipodophyllotoxin for podophyllotoxin, one obtains thecorresponding 4′-demethylepipodophyllotoxin compound.

C. 4′-Phosphate ester of 4′-demethylepipodophyllotoxin-4-O-ester ofN—BOC-1,2,3,4-Tetrahydroisoquinoline-3-carboxylic acid

The compound prepared in part B of this Example is converted to thecorresponding 4′-phosphate ester as follows: A suspension of 50% NaH inmineral oil and the 4′-demethylepipodophyllotoxin-4-O-ester of Part B ofthis Example in DMF is stirred for 30 minutes at room temperature. Afterthe mixture is cooled to 0° C., a solution ofdibenzylphosphorochloridate in toluene is added drop by drop. Thesolution is stirred at room temperature for 15 minutes and then dilutedwith cold water and extracted with ether. The ether solution is washedwith water, dried, evaporated under reduced pressure to give the4′-dibenzylphosphate derivative of the compound of part B. A solution ofthe dibenzylphosphate derivative in 85% ethanol is hydrogenated in aParr apparatus in the presence of 10% Pd supported on carbon. Aftertheoretical absorption of hydrogen, the catalyst is filtered, washedwith cold water and suspended in 2N NH₄OH at 50° C. The suspension isfiltered, washed with water and concentrated at reduced pressure at 50°C. The solution is then filtered and acidified with HCl to give thetitle compound.

Example 24 A. Podophyllotoxin-4-O-ester ofN—BOC-erythro-D-β-Menthylphenylalanine [000307]

The mixture of podophyllotoxin (20 mg, 0.048 mmol),N—BOC-erythro-D-β-Menthylphenylalanine (28 mg, 0.096 mmol), EDCI (25 mg,0.13 mmol), DMAP (2 mg, 0.02 mmol) and dichloromethane (3 ml) werestirred in the room temperature for 20 h, then dichloromethane (20 ml)was added to the solution. Organic layer was washed with water (20 ml),saturated NaHCO₃ aqueous solution (10 ml) and brine (20 ml), and thendried over MgSO₄. After the solvent was removed under reduced pressure,the resulting liquid was separated by column chromatography (eluent:ethyl acetate and petroleum ether) to afford 23 mgPodophyllotoxin-4-O-ester of N—BOC-erythro-D-β-Menthylphenylalanine

The chemical structure analysis was performed by ¹HNMR (CDCl₃, 600 MHz):

B. 4′-demethylepipodophyllotoxin-4-O-ester ofN—BOC-erythro-D-β-Menthylphenylalanine

By following the procedure of Part A of this Example, but substituting4′-demethylepipodophyllotoxin for podophyllotoxin, one obtains thecorresponding 4′-demethylepipodophyllotoxin compound.

C. 4′-Phosphate ester of 4′-demethylepipodophyllotoxin-4-O-ester ofN—BOC-erythro-D-β-Menthylphenylalanine

The compound prepared in part B of this Example is converted to thecorresponding 4′-phosphate ester as follows: A suspension of 50% NaH inmineral oil and the 4′-demethylepipodophyllotoxin-4-O-ester of Part B ofthis Example in DMF is stirred for 30 minutes at room temperature. Afterthe mixture is cooled to 0° C., a solution ofdibenzylphosphorochloridate in toluene is added drop by drop. Thesolution is stirred at room temperature for 15 minutes and then dilutedwith cold water and extracted with ether. The ether solution is washedwith water, dried, evaporated under reduced pressure to give the4′-dibenzylphosphate derivative of the compound of part B. A solution ofthe dibenzylphosphate derivative in 85% ethanol is hydrogenated in aParr apparatus in the presence of 10% Pd supported on carbon. Aftertheoretical absorption of hydrogen, the catalyst is filtered, washedwith cold water and suspended in 2N NH₄OH at 50° C. The suspension isfiltered, washed with water and concentrated at reduced pressure at 50°C. The solution is then filtered and acidified with HCl to give thetitle compound.

Example 25 A. Podophyllotoxin-4-O-ester of camptothecin-20-O-ester of4-carboxylicphenoxyacetic acid [000801]

The mixture of podophyllotoxin (30 mg, 0.072 mmol),camptothecin-20-O-ester of 4-carboxylicphenoxyacetic acid (15 mg, 0.028mmol—see U.S. Pat. No. 6,350,756), EDCI (20 mg, 0.10 mmol), DMAP (2 mg,0.02 mmol) and dichloromethane (3 ml) were stirred in the roomtemperature for 20 h, then dichloromethane (20 ml) was added to thesolution. Organic layer was washed with water (20 ml), saturated NaHCO₃aqueous solution (10 ml) and brine (20 ml), and then dried over MgSO₄.After the solvent was removed under reduced pressure, the resultingliquid was separated by column chromatography (eluent: ethyl acetate andpetroleum ether) to afford 5.0 mg Podophyllotoxin-4-O-ester ofcamptothecin-20-O-ester of 4-carboxylicphenoxyacetic acid.

The chemical structure analysis was performed by ¹HNMR (CDCl₃, 600 MHz):

B. 4′-demethylepipodophyllotoxin-4-O-ester of camptothecin-20-O-ester of4-carboxylicphenoxyacetic acid

By following the procedure of Part A of this Example, but substituting4′-demethylepipodophyllotoxin for podophyllotoxin, one obtains thecorresponding 4′-demethylepipodophyllotoxin compound.

C. 4′-Phosphate ester of 4′-demethylepipodophyllotoxin-4-O-ester ofcamptothecin-20-O-ester of 4-carboxylicphenoxyacetic acid

The compound prepared in part B of this Example is converted to thecorresponding 4′-phosphate ester as follows: A suspension of 50% NaH inmineral oil and the 4′-demethylepipodophyllotoxin-4-O-ester of Part B ofthis Example in DMF is stirred for 30 minutes at room temperature. Afterthe mixture is cooled to 0° C., a solution ofdibenzylphosphorochloridate in toluene is added drop by drop. Thesolution is stirred at room temperature for 15 minutes and then dilutedwith cold water and extracted with ether. The ether solution is washedwith water, dried, evaporated under reduced pressure to give the4′-dibenzylphosphate derivative of the compound of part B. A solution ofthe dibenzylphosphate derivative in 85% ethanol is hydrogenated in aParr apparatus in the presence of 10% Pd supported on carbon. Aftertheoretical absorption of hydrogen, the catalyst is filtered, washedwith cold water and suspended in 2N NH₄OH at 50° C. The suspension isfiltered, washed with water and concentrated at reduced pressure at 50°C. The solution is then filtered and acidified with HCl to give thetitle compound.

D. Podophyllotoxin-4-O-ester of camptothecin derivative-20-O-ester of4-carboxylicphenoxyacetic acid

By following the methods described above and substituting camptothecinwith a camptothecin derivative disclosed in U.S. Pat. No. 6,350,756,those skilled in the art will be able to prepare a compound of thisinvention analogous to compounds described in A, B, and C of thisexample, which has a 20-O-ester of a camptothecin derivative.

Example 26 A. Bis(Podophyllotoxin-4-O-ester) of 3,5-Pyridinedicarboxylicacid [000330]

The mixture of podophyllotoxin (36 mg, 0.072 mmol),3,5-Pyridinedicarboxylic acid (10 mg, 0.036 mmol), EDCI (25 mg, 0.13mmol), DMAP (2 mg, 0.02 mmol) and dichloromethane (3 ml) were stirred inthe room temperature for 20 h, then dichloromethane was added to thesolution. Organic layer was washed with water, saturated NaHCO₃ aqueoussolution and brine, and then dried over MgSO₄. After the solvent wasremoved under reduced pressure, the resulting liquid was separated bycolumn chromatography (eluent: ethyl acetate and petroleum ether) toafford 20 mg of bis(podophyllotoxin-4-O-ester of3,5-pyridinedicarboxylic acid.

The chemical structure analysis was performed by ¹HNMR (CDCl₃, 600 MHz):

B. Bis(4′-demethylepipodophyllotoxin-4-O-ester) of3,5-Pyridinedicarboxylic acid

By following the procedure of Part A of this Example, but substituting4′-demethylepipodophyllotoxin for podophyllotoxin, one obtains thecorresponding 4′-demethylepipodophyllotoxin compound.

C. Bis(4′-Phosphate ester of 4′-demethylepipodophyllotoxin-4-O-ester) of3,5-Pyridinedicarboxylic acid

The compound prepared in part B of this Example is converted to thecorresponding 4′-phosphate ester as follows: A suspension of 50% NaH inmineral oil and the 4′-demethylepipodophyllotoxin-4-O-ester of Part B ofthis Example in DMF is stirred for 30 minutes at room temperature. Afterthe mixture is cooled to 0° C., a solution ofdibenzylphosphorochloridate in toluene is added drop by drop. Thesolution is stirred at room temperature for 15 minutes and then dilutedwith cold water and extracted with ether. The ether solution is washedwith water, dried, evaporated under reduced pressure to give the4′-dibenzylphosphate derivative of the compound of part B. A solution ofthe dibenzylphosphate derivative in 85% ethanol is hydrogenated in aParr apparatus in the presence of 10% Pd supported on carbon. Aftertheoretical absorption of hydrogen, the catalyst is filtered, washedwith cold water and suspended in 2N NH₄OH at 50° C. The suspension isfiltered, washed with water and concentrated at reduced pressure at 50°C. The solution is then filtered and acidified with HCl to give thetitle compound.

Example 27

This example provides directions for growing cells and testing compoundsof the invention for their effect on the growth of the cells. All cellswere purchased from DCTDC Tumor Repository, NCI, NIH.

Cell Colony Formation Assay

Four hundred cells (HCT 116, PC-3) or five hundred cells (VM46) wereplated in 60 mm Petri dishes containing 2.7 ml of medium (modifiedMcCoy's 5a medium) containing 10% fetal bovine serum and 100 units/mlpenicillin and 100 mg/ml streptomycin. The cells were incubated in a CO₂incubator at 37° C. for 5 hours for attachment to the bottom of Petridishes. Drugs were made fresh in medium at ten times the finalconcentration, and then 0.3 ml of this stock solution was added to the2.7 ml of medium in the dish. The cells were then incubated with drugsfor 72 hours at 37° C. At the end of the incubation the drug-containingmedia were decanted, the dishes were rinsed with 4 ml of Hank's BalanceSalt Solution (HBSS), 5 ml of fresh medium was added, and the disheswere returned to the incubator for colony formation. The cell colonieswere counted using colony counter after incubation for 7 days for HCT116cells and PC-3 cells and 8 days for VM46 cells, respectively. Cellsurvival (%) was calculated, as shown in Table I below for HCT 116cells.

Values of ID50 (the drug concentration producing 50% inhibition ofcolony formation) may be determined for each tested compound. Thedirections described in this example may be used in other cells, such asDU-145. TABLE I This table provides results of in vitro efficacy testsperformed in example 16 for the cell line HCT116. In Vivo EfficacySurviving days after In Vivo Toxicity treatment of MTG-B In VitroEfficacy: Survival (%) of nontoxic dose mouse mammary HCT116 (ip, mg/kg)in adenocarcinoma in Compounds 1000 nM 100 nM 10 nM 1 nM C3H/Hcj miceC3H/Hej mice T/C % Etoposide 100 000121 0 94.03 000201 0 93.24 000125 090.34 000202 0 0 81.68 000203 0 89.76 000124 0 0 83.22 000215 0 0 92.46000218 0 44.28 000222 0 1.46 100 000301 0 84.08 000302 0 100 0003061  00 100 100 (70) 13 217 000307 0 93.49 0003132  0 97.65 000317 0 0 99.49000320 0 0 100 000323 0 0 90.86 000324 0 0 100 000329 0 0 100 000330 0 0100 000331 0 62.56 000405 0 0 90.45 000614 0 0 84.99 000615 0 0 100000622 0 0 98.48 000801 0 0 0 99.83

Example 28

This example provides directions for performing in vivo toxicity testsof the compounds of the invention on C3H/HeJ mice.

Acute toxicities of the compounds of this invention are evaluated onC3H/HeJ mice (body weight 18-22 g). The MTD40 (maximum tolerated dose atday 40) values are determined by the standard procedure described by Gadand Chengelis (see, for example, “Acute Toxicology Testing,” 2^(nd) Ed.,Shayne O. Gad and Christopher P. Chengelis, pp. 186-195 (AcademicPress).) In the consecutive type studies, 2 mice are dosed at low andmoderate doses of 40 and 100 mg/kg. If no severe and irreversibletoxicity (euthanasia is required) occurs at these doses, a new pair ofanimals is initiated at 180 mg/kg, which is 1.8 times higher than 100mg/kg. Sequential dosages (about 3 doses on 3 pairs of animals, i.e. 2mice for each drug dose) are increased by a factor of 1.8 until severeand irreversible toxicity (euthanasia is required) occurred. Thenanother pair of animals is initiated at the highest nonlethal dosage,and successive dosages were increased by a factor of 1.15. The result ofthis exercise is two dosages, one apparently nonlethal and the otherlethal if severe and irreversible toxicity occurs and euthanasia isrequired, separated by a factor of 1.15. Six mice are dosed at eachdosage. If no severe and irreversible toxicity occurs at the lowerdosage and at least one with severe and irreversible toxicity occurs atthe higher dose, then the lower dose is considered to be the MTD. Thecompounds of this invention are administered to C3H/HeJ mice byintraperitoneal injection. Drug toxicity is evaluated on mice checkeddaily for 45 days. The toxicity parameters reported will be the MTD40.The MTD is defined as the highest dose causing no severe irreversibletoxicity in one treatment group, but at least one animal exhibitingsevere and irreversible toxicity and being euthanized at the next higherdose. The acute toxicity of the podophyllotoxin-4-O-ester ofthymine-1-acetic acid (compound 3061, described in Example 16) is shownabove in Table 1.

Example 29

This example provides directions for performing in vivo efficacy testsof the compounds of the invention on C3H/HeJ mice bearing MTG-B tumors.

Studies on the compounds of this invention are performed on C3H/HeJ micebearing MTG-B tumors. The tumors grow exponentially followingimplantation into the flanks of the mice and reached a diameter of 8 mm(268.08 mm³) by day 7 to 10. Treatment is initiated at that time, withthe first day of treatment designated as day 0 for calculation andplots. The mice are injected i.p. with three drug dose levels (⅓, ½,1×MTD) using both a single injection and the schedule of Q2D×3 (every 2days for a total of 3 treatments at ⅓ MTD). Control groups of micebearing 8 mm diameter tumors are treated with vehicle alone. After drugtreatment, the mice are observed twice a day. When a tumor reaches 1.5g, the mouse bearing the tumor is euthanized. Surviving days measuredfrom day 0 for mice treated with anticancer drugs (T) and surviving daysmeasured from day 0 for control mice (C) are recorded. Tumor growthinhibition values (T/C %) are calculated using the formula T/C%=(surviving days of mice treated with an anticancer drug T/survivingdays of control mice C)×100%.

Tumor sizes may be measured by caliper every day. Daily measurement (mm)of solid tumor (length L and width W) in two dimensions is used tocalculate the tumor weight [tumor weight=(length×width²)/2] based on theinterchangeable value of 1 mm³=1 mg. Tumor growth delay (T−C value) isdetermined by calculation of the median time (in days) required for thetreatment group and control group tumors to reach 1,000 mg. Tumordoubling time (Td) is measured, and tumor cell kill is calculated by theformula of log cell kill=(T−C value)/(3.32×Td). Regression effects aftertreatment may be observed and recorded (a complete regression: aregression below limit of palpation; a partial regression: a regressionof more than 50% reduction in tumor mass).

Generally, the survival time of the control mice is six (6) days. Aratio of the extra days of survival of mice treated with the compoundsof the invention (compared to control) to the extra days of survival ofmice treated with taxol (compared to control), can be calculated. Forexample, if the mice survived 18 days as compared to 9 days fortaxol-treated mice, the CD/Taxol ratio would be 18-6/9-6=12/3=4. The invivo efficacy of the podophyllotoxin-4-O-ester of thymine-1-acetic acid(compound 3061, described in Example 16) is shown in Table 1 below.

Example 30

This example provides guidance for determining the inhibition oftopoisomerase II. This procedure is a modification of a publishedprocedure for the P4 knotted DNA unknotting reaction found at J. Biol.Chem. 1983, 258, 8413. A more recent publication can be found at J. Med.Chem. 1989, Vol. 32, No. 3 at page 608. A reaction mixture (20 μL),which contains 50 mM HEPES pH 6.7, 50 mM KCl, 100 mM NaCl, 0.1 mM EDTA,10 mM HgCl₂, 0.1 mM ATP, 50 μg/mL bovine serum albumin, 0.26 μg P4knotted DNA, and enzyme, is incubated with or without a compound of theinvention.

The reaction mixture is incubated at 37° C. for 30 min and terminated byadding a stop solution (2% sodium dodecyl sulfate, 20% glycerol, 0.05%bromphenol blue). These samples are loaded onto a 1% agarose gel andelectrophoresed at 50 V overnight with an electrophoresis buffer thatcontains 90 mM Tris-boric acid, pH 8.3, and 2.5 mM EDTA. At completion,the gel is stained in 0.5 μg/mL of ethidium bromide. Then a photographis taken of the DNA bands visualized with fluorescence induced by along-wavelength UV lamp. The data is reported.

1. A compound represented by the formula:

wherein R is C(O)—(CH₂)_(m)—X—R₁, wherein m is 0, X is a covalent bond,and R₁ is

R₂ is hydrogen, PO₃H₂ or PO(OR₃)₂ where R₃ is benzyl; R₂₂ is hydrogen,halo, lower alkyl, lower alkoxy, hydroxy, R₄₀C(O)O, cyano, nitro, amino,halogenated lower alkyl, halogenated lower alkoxy, hydroxycarbonyl,formyl, lower alkoxycarbonyl, tri lower alkylsilyl, loweralkylcarbonyloxy, lower alkylcarbonylamino, loweralkylcarbonyloxymethyl, substituted vinyl, 1-hydroxy-2-nitroethyl,alkoxycarbonylethyl, aminocarbonyl, mono- or di-alkylcarbonyl,alkylcarbonylmethyl, benzoylmethyl, benzylcarbonyloxymethyl, or mono- ordi lower alkoxymethyl; R₂₃ is hydrogen, halo, lower alkyl, lower alkoxy,hydroxy, R₄₀C(O)O, cyano, nitro, amino, halogenated lower alkyl,halogenated lower alkoxy, hyroxycarbonyl, formyl, lower alkoxycarbonyl,CH₂NR₂₇R₂₈ (where each of R₂₇ and R₂₈ is independently H—, alkyl of 1-6carbons, optionally substituted phenyl, hydroxy lower alkyl, amino loweralkyl, or mono- or dialkylamino lower alkyl, or R₂₇ and R₂₈ takentogether with —N— represent a cyclic amino-), CH₂R₂₉ (where R₂₉ is loweralkoxy, CN, amino lower alkoxy, mono- or di-lower alkylamino loweralkoxy, lower alkylthio, amino lower alkylthio, or mono- or di-loweralkylamino lower alkylthio), or NR₃₀R₃₁ (where each of R₃₀ and R₃₁ isindependently hydrogen, lower alkyl, phenyl, hydroxy lower alkyl, aminolower alkyl, or mono- or di-lower alkyl, or R₃₀ and R₃₁ taken togetherwith —N— represent a cyclic amino), dialkylamino alkyl, loweralkylcarbonyloxy, or lower alkylcarbonylamino; R₂₄ is hydrogen, halo,lower alkyl, lower alkoxy, hydroxy, R₄₀C(O)O, cyano, nitro, amino, aminolower alkyl, halogenated lower alkyl, halogenated lower alkoxy,hydroxycarbonyl, formyl, lower alkoxycarbonyl, carbamoyloxy, loweralkylcarbonyloxy, or lower alkylcarbonylamino, or R₂₄ together with R₂₅is methylenedioxy; R₂₅ is hydrogen, halo, lower alkyl, lower alkoxy,hydroxy, R₄₀C(O)O, cyano, nitro, amino, halogenated lower alkyl,halogenated lower alkoxy, hydroxycarbonyl, formyl, lower alkoxycarbonyl,lower alkylcarbonyloxy, or lower alkylcarbonylamino; and R₂₆ ishydrogen, halo, lower alkyl, lower alkoxy, hydroxy, R₄₀C(O)O, cyano,nitro, amino, halogenated lower alkyl, halogenated lower alkoxy,hydroxycarbonyl, formyl, lower alkoxycarbonyl, lower alkylcarbonyloxy,or lower alkylcarbonylamino; R₄₀ is R₄₁—O—(CH₂)_(s)—; s is an integer of1-10; R₄₁ is lower alkyl; phenyl optionally substituted with from one tofive substituents independently selected from the group consisting ofhalo, lower alkyl, lower alkoxy, hydroxy, cyano, nitro, amino,halogenated lower alkyl, halogenated lower alkoxy, formyl, lower alkylcarbonyl, hydroxycarbonyl, lower alkylcarbonyloxy, benzyloxy, optionallysubstituted piperazino, lower alkoxycarbonyl, and loweralkylcarbonylamino; cycloalkyl of 3-7 carbons, optionally substitutedwith one to five substituents independently selected from the groupconsisting of halo, lower alkyl, lower alkoxy, hydroxy, cyano, nitro,amino, halogenated lower alkyl, halogenated lower alkoxy,hydroxycarbonyl, lower alkoxycarbonyl, lower alkylcarbonyloxy, and loweralkylcarbonylamino; a fused, 2-, 3-, or 4-ring heterocyclic systemoptionally substituted with one to five substituents independentlyselected from the group consisting of halo, lower alkyl, lower alkoxy,hydroxy, cyano, nitro, amino, halogenated lower alkyl, halogenated loweralkoxy, hydroxycarbonyl, lower alkoxycarbonyl, lower alkylcarbonyloxy,and lower alkylcarbonylamino; 1- or 2-naphthyl optionally substitutedwith from one to four substituents independently selected from the groupconsisting of halo, lower alkyl, lower alkoxy, hydroxy, cyano, nitro,amino, halogenated lower alkyl, halogenated lower alkoxy,hydroxycarbonyl, lower alkoxycarbonyl, lower alkylcarbonyloxy, and loweralkylcarbonylamino; and a 5 or 6 membered heterocyclic ring containingone or two nitrogen atoms, which ring is optionally substituted with oneor two substituents selected from the group consisting of halo, loweralkyl, lower alkoxy, hydroxy, cyano, nitro, amino, halogenated loweralkyl, halogenated lower alkoxy, hydroxycarbonyl, lower alkoxycarbonyl,lower alkylcarbonyloxy, and lower alkylcarbonylamino; wherein the wavyline represents the point of connection to X.
 2. The compound of claim1, wherein R₂₂ is hydrogen; R₂₃ is CH₂NR₂₇R₂₈ (where each of R₂₇ and R₂₈is independently H—, alkyl of 1-6 carbons, optionally substitutedphenyl, hydroxy lower alkyl, amino lower alkyl, or mono- or dialkylaminolower alkyl, or R₂₇ and R₂₈ taken together with —N— represent a cyclicamino-), NR₃₀R₃₁ (where each of R₃₀ and R₃₁ is independently hydrogen,lower alkyl, phenyl, hydroxy lower alkyl, amino lower alkyl, or mono- ordi-lower alkyl, or R₃₀ and R₃₁ taken together with —N— represent acyclic amino), or dialkylamino alkyl; R₂₄ is lower alkoxy, hydroxy,halogenated lower alkyl, halogenated lower alkoxy, hydroxycarbonyl,formyl, lower alkoxycarbonyl, carbamoyloxy, lower alkylcarbonyloxy, orR₂₄ together with R₂₅ is methylenedioxy; R₂₅ is hydrogen, or togetherwith R₂₄ is methylenedioxy; R₂₆ is hydrogen.
 3. The compound of claim 2,wherein R₂₃ is CH₂NR₂₇R₂₈ (where each of R₂₇ and R₂₈ is lower alkyl),R₂₅ is hydrogen, and R₂₄ is hydroxy, alkoxy or alkylcarbonyloxy.
 4. Thecompound of claim 3, wherein R₂₃ is CH₂N(CH₃)₂ and R₂₄ is hydroxy. 5.The compound of claim 1, wherein R₂₂ is hydrogen, lower alkyl, orhalogenated lower alkyl; R₂₃ is hydrogen or lower alkyl; R₂₄ is loweralkoxy, hydroxy, halogenated lower alkoxy, carbamoyloxy, loweralkylcarbonyloxy, or R₂₄ together with R₂₅ is methylenedioxy; R₂₅ ishydrogen, or together with R₂₄ is methylenedioxy; and R₂₆ is hydrogen.6. The compound of claim 5, wherein R₂₃ is hydrogen, R₂₅ is hydrogen,and R₂₄ is carbamoyloxy.
 7. The compound of claim 5, wherein R₂₂ islower alkyl and R₂₄ is 4-(1-piperidino)-1-piperidinocarbonyloxy.
 8. Thecompound of claim 7, wherein R₂₂ is ethyl.
 9. The compound of claim 1,wherein R₂₂ is lower alkyl; R₂₃ is hydrogen; R₂₄ is hydroxy, loweralkoxy, halogenated lower alkoxy, hydroxycarbonyl, formyl, loweralkoxycarbonyl, carbamoyloxy, lower alkylcarbonyloxy; R₂₅ is hydrogen;and R₂₆ is hydrogen.
 10. The compound of claim 9, wherein R₂₂ is ethyland R₂₄ is hydroxy.
 11. The compound of claim 1, wherein each of R₂₂,R₂₄, R₂₅, and R₂₆ is hydrogen; and R₂₃ is amino or nitro.
 12. Thecompound of claim 11, wherein R₂₃ is amino.
 13. The compound of claim12, wherein R₂₃ is nitro.
 14. The compound of claim 1, wherein R₂₂ istri-lower alkylsilyl; R₂₃ is hydrogen; R₂₄ is hydroxy, lower alkoxy,halogenated lower alkoxy, hydroxycarbonyl, formyl, lower alkoxycarbonyl,carbamoyloxy, lower alkylcarbonyloxy; R₂₅ is hydrogen; and R₂₆ ishydrogen.
 15. The compound of 14, wherein R₂₂ is t-butyldimethylsilyland R₂₄ is hydroxy.
 16. A compound of claim 1, selected from:

or a pharmaceutically acceptable salt thereof.
 17. The compound of claim1 in combination with a pharmaceutically-acceptable excipient to form apharmaceutical composition.
 18. The pharmaceutical composition of claim17, which is in the form of a liposomal composition.
 19. A method fortreating cancer in a patient, which method comprises administering atherapeutically effective amount of a compound of claim 1 to thepatient.
 20. A compound represented by the formula:

wherein R is C(O)—(CH₂)_(m)—X—R₁, wherein m is 0-10, X is S, O N or acovalent bond, and R₁ is optionally substituted phenyl, optionallysubstituted cycloalkyl having 3 to 7 carbons forming the ring,optionally substituted fused 2-, 3-, or 4-ring heterocycle, optionallysubstituted 1- or 2-naphthyl, optionally substituted anthraquinone, orhemisuccinic acid; with the proviso that when m is 0 and X is a bond, R₁cannot be phenyl or substituted phenyl; when X is a bond and R₁ isphenyl, m cannot be 2 and when X is O, m cannot be
 1. 21. The compoundof claim 20, wherein m is 1-10 and R₁ is phenyl substituted with one tofive substituents independently selected from halo, lower alkyl,hydroxy, lower alkoxy, cyano, nitro, amino, lower alkylamino,halogenated lower alkylamino, halogenated lower alkyl, halogenated loweralkoxy, carbonyl, hydroxycarbonyl, lower alkylcarbonyloxy, benzyloxy,optionally substituted 5 or 6 membered heterocyclic ring, an imide ring,lower alkoxycarbonyl, and lower alkylcarbonylamino.
 22. The compound ofclaim 21, wherein m is 1 to 3 and X is S or O.
 23. The compound of claim22, wherein m is 1, X is O and R₁ is phenyl substituted with 1, 2, or 3substituents independently chosen from, halo, methyl, methoxy, NO₂,trifluoromethyl, and carbonyl.
 24. The compound of claim 23, wherein R₁is phenyl substituted with one or two halo substituents.
 25. Thecompound of claim 23, wherein R₁ is phenyl substituted with a methylsubstituent.
 26. The compound of claim 20, wherein m is 1 and R₁ isoptionally substituted cycloalkyl having 3 to 7 carbons forming thering, optionally substituted fused 2-, 3-, or 4-ring heterocycle,optionally substituted 5- or 6-membered heterocycle, or optionallysubstituted anthraquinone.
 27. The compound of claim 20, wherein m is 0to 3; and X is oxygen.
 28. The compound of claim 20, wherein m is 0 to3; and X a covalent bond.
 29. The compound of claim 28, wherein R₁ is afused heterocyclic ring system.
 30. The compound of claim 29, wherein R₁is optionally substituted quinolin-4-yl.
 31. The compound of claim 30,wherein R₁ is 2-phenylquinolin-4-yl.
 32. The compound of claim 29,wherein the fused heterocyclic ring system is chromon-2-yl.
 33. Thecompound of claim 28, wherein R₁ is a fused carbocyclic system.
 34. Thecompound of claim 33, wherein R₁ is anthraquinon-1-yl.
 35. The compoundof claim 20 in combination with a pharmaceutically-acceptable excipientto form a pharmaceutical composition.
 36. The pharmaceutical compositionof claim 35, which is in the form of a liposomal composition.
 37. Amethod for treating cancer in a patient, which method comprisesadministering a therapeutically effective amount of a compound of claim20 to the patient.
 38. A compound represented by the formula:

wherein R is C(O)—(CH₂)_(m)—X—R₁, wherein m is 0-10, X is S, O, N or acovalent bond, and R₁ is optionally substituted phenyl, optionallysubstituted cycloalkyl having 3 to 7 carbons forming the ring,optionally substituted fused 2-, 3-, or 4-ring heterocycle, optionallysubstituted 1- or 2-naphthyl, optionally substituted 5- or 6-memberedheterocycle, optionally substituted anthraquinone, or hemisuccinic acid,and R₂ is hydrogen, PO₃H₂ or PO(OR₃)₂ where R₃ is benzyl.
 39. Thecompound of claim 38 wherein R₂ is hydrogen or PO₃H₂.
 40. The compoundof claim 39, wherein m is 1-10 and R₁ is phenyl substituted with one tofive substituents independently selected from halo, lower alkyl,hydroxy, lower alkoxy, cyano, nitro, amino, lower alkylamino,halogenated lower alkylamino, halogenated lower alkyl, halogenated loweralkoxy, carbonyl, hydroxycarbonyl, lower alkylcarbonyloxy, benzyloxy,optionally substituted 5 or 6 membered heterocyclic ring, an imide ring,lower alkoxycarbonyl, and lower alkylcarbonylamino.
 41. The compound ofclaim 40, wherein m is 1 to 3 and X is S or O.
 42. The compound of claim41, wherein m is 1, X is O and R₁ is phenyl substituted with 1, 2, or 3substituents independently chosen from, halo, methyl, methoxy, NO₂,trifluoromethyl, and carbonyl.
 43. The compound of claim 42, wherein R₁is phenyl substituted with one or two halo substituents.
 44. Thecompound of claim 42, wherein R₁ is phenyl substituted with a methylsubstituent.
 45. The compound of claim 38, wherein m is 1 and R₁ isoptionally substituted cycloalkyl having 3 to 7 carbons forming thering, optionally substituted fused 2-, 3-, or 4-ring heterocycle,optionally substituted 5- or 6-membered heterocycle, or optionallysubstituted anthraquinone.
 46. The compound of claim 38, wherein m is 0to 3; and X is oxygen.
 47. The compound of claim 38, wherein m is 0 to3; and X is a covalent bond.
 48. The compound of claim 47, wherein R₁ isan optionally substituted 5-membered heterocycle with an oxygen or oneor two nitrogens in the ring.
 49. The compound of claim 48, wherein R₁is optionally substituted furan-2-yl.
 50. The compound of claim 49,wherein R₁ is 5-nitrofuran-2-yl.
 51. The compound of claim 47, whereinR₁ is an optionally substituted 6-membered heterocycle with one or twonitrogens in the ring.
 52. The compound of claim 51, wherein the6-membered heterocycle is pyridine-3-yl, thymin-1-yl, or piperazin-1-yl.53. The compound of claim 47, wherein R₁ is a fused heterocyclic ringsystem.
 54. The compound of claim 53, wherein R₁ is optionallysubstituted quinolin-4-yl.
 55. The compound of claim 54, wherein R₁ is2-phenylquinolin-4-yl.
 56. The compound of claim 53, wherein the fusedheterocyclic ring system is chromon-2-yl.
 57. The compound of claim 47,wherein R₁ is a fused carbocyclic system.
 58. The compound of claim 57,wherein R₁ is anthraquinon-1-yl.
 59. The compound of claim 38 incombination with a pharmaceutically-acceptable excipient to form apharmaceutical composition.
 60. The pharmaceutical composition of claim59, which is in the form of a liposomal composition.
 61. A method fortreating cancer in a patient, which method comprises administering atherapeutically effective amount of a compound of claim 38 to thepatient.
 62. A compound selected from compounds 000121, 000201, 000125,000203, 000124, 000215, 000218, 000222, 000301, 000302, 000307, 0003132,000317, 000320, 000323, 000324, 000329, 000330, 000331, 000405, 000614,000615, and
 000622. 63. A compound represented by the formulaA-R₅—B wherein each of A and B independently is represented by theradical

wherein R₂ is hydrogen, PO₃H₂ or PO(OR₃)₂ where R₃ is benzyl and R₅ is adicarboxy linker.
 64. The compound of claim 63, wherein R₅ is2,4-dicarboxy-5-nitrophenyl.
 65. The compound of claim 63, wherein R₅ is3,5-dicarboxy-pyridine.
 66. The compound of claim 64, wherein A and Bare the same.
 67. The compound of claim 64, wherein A and B aredifferent.
 68. The compound of claim 63 in combination with apharmaceutically acceptable excipient to form a pharmaceuticalcomposition.
 69. A method for treating cancer in a patient, which methodcomprises administering a therapeutically effective amount of a compoundof claim 63 to the patient.